Class 




Copyright N° 



COPYRIGHT DEPOSIT. 



MULLER'S 
SERODIAGNOSTIC 
METHODS 

AUTHORIZED TRANSLATION FROM 
THE THIRD GERMAN EDITION 

BY 

ROSS C. WHITMAN, B.A., M.D. 

PROFESSOR OF PATHOLOGY, UNIVERSITY OF COLORADO SCHOOL OF MEDICINE 

WITH 7 ILLUSTRATIONS IN TEXT 




PHILADELPHIA & LONDON 
J. B. LIPPINCOTT COMPANY 



COPYRIGHT, 1913 
BY J. B. LIPPINCOTT COMPANY 



Electrotyped and Printed by J. B. Lippincott Company 
The Washington Square Press, Philadelphia, U. S. A. 



©CI.A346430 

^0/ 



TRANSLATOR'S PREFACE 

Professor Miiller's remarkably clear presen- 
tation of the complicated methods embraced under 
the term " Sero-diagnosis," cannot fail to find a 
welcome among the rapidly increasing number 
who are entering this field of work in America. 
The Translator has added the following methods, 
which have appeared since the German edition was 
published — the Hermann and Perutz method for 
the diagnosis of syphilis; the meiostagmin reac- 
tion of Ascoli and Izar; the cancer diagnosis of 
Freund and Kaminer; the cobra venom reaction 
in syphilis of Weil; the cobra venom reaction in 
cancer of Kraus, Graff, and Eanzi; the comple- 
ment deviation tests of von Dungern, Schwartz 
and McNeil, and of Hammer, for cancer, gonor- 
rhoea and tuberculosis respectively. Certain other 
additions of a minor character, and certain sug- 
gestions offered by the experience of the Trans- 
lator, have been included in brackets. "With these 
exceptions the translation follows closely the Ger- 
man original. The Noguchi modification of the 
Wassermann Reaction has been omitted at the 
request of its author. All apparatus and equip- 
ment mentioned in the text, which is available on 
the market, can be obtained from the Kny-Scherer 

iii 



iv 



TRANSLATOR'S PREFACE. 



Company, New York, and from other dealers in 
laboratory supplies. I desire, also, to express my 
sincere appreciation of the kindness of the pub- 
lishers in permitting me to make changes in, and 
substantial additions to, the text, down to the very 
moment of going to press. 

Eoss C Whitman. 

Denver, February 15, 1913. 



PREFACE TO THE SECOND 
EDITION 

This collection of the more important methods 
of serum diagnosis, originally made for the use 
of my students, may perhaps be welcome to many 
who dislike the labor of collecting the data scat- 
tered through the literature. In accordance with 
the purely practical purpose which suggested the 
work, theoretical considerations have been entirely 
omitted. The chief emphasis has, on the contrary, 
been laid on making the description of the various 
methods as exact as possible, and especially on 
giving a complete and detailed list of the reagents 
and apparatus required for each test. With a 
definite purpose in view, certain methods have 
been included, the permanent worth of which has 
not yet been demonstrated ; for it is precisely these 
methods which deserve an especially thorough 
testing out, under the most varied conditions. 
Additions to the second edition are: a chapter 
on the preservation of sera ; the determination of 
meat adulteration according to the method of 
Uhlenhuth, Weidanz, and Wedemann; and the 
floccule forming reactions, especially floccule 
forming tests for syphilis with lecithin, and with 
sodium glycocholate, and Klausner's reaction; 
Moro's alexin test; and finally, of the various 



vi PREFACE TO THE SECOND EDITION. 



modifications of the Wassermann reaction, I 
have given those of Tage, Hahne, Bauer, and 
Tschernobugon;. 

Absolute mastery of sero-diagnostic methods 
can, of course, only be acquired by practice and 
experience. I shall rejoice if this booklet serves 
to lighten the labor of the beginner in this field. 

Paul Th. Muller. 



PREFACE TO THE THIRD 
EDITION 

The third edition of this booklet, coming as it 
does, so soon after its predecessor, can of course, 
differ but slightly from the latter. The following 
methods have been added : the improved and sim- 
plified test for syphilis of Stern, the Much- 
Holzmann "Psycho-reaction" ; and the estimation 
of the antitryptic power of the blood according to 
the method of Miiller and Jochmann (modified by 
Marcus), and according to the method of 
Bergmann and Meyer. The entirely proper wish 
of one of the book's critics, that the methods 
which have not justified themselves, might be 
mercilessly eliminated from future editions, can- 
not yet be complied with, since the experience 
needed to determine the value of such methods is 
still all too slight. 

Paul Th. Muller. 



vii 



CONTENTS 



Methods of Injecting Animals page 

Intraperitoneal Injection of Rabbits to Produce Im- 
mune Serum 1 

Intravenous Injection of Rabbits 1 

Obtaining Blood from Animals 

Drawing Blood from Ear Vein of Rabbits to Obtain Small 
Amounts of Serum 2 

Drawing Bix>od from Rabbits to Obtain Large Amounts 
of Serum 4 

Obtaining Blood from Other Animals 6 

Obtaining Small Amounts of Human Blood 

Proscher'sMethod 7 

czaplewski-schottelius method 9 

Obtaining Larger Amounts of Human Blood 

Obtaining Placental Blood 10 

Use of Heurteloup or Wet Cup 11 

Venipuncture or Phlebotomy 11 

Use of Cadaveric Blood 11 

Obtaining Defibrinated Blood or Blood-cells, , 11 

Preservation of Sera 
Preservation of Normal Sera 13 

Preservation of Immune Sera 

Preservation in Fluid Condition by Means of Antisep- 
tics 14 



X 



CONTENTS. 



Preservation in Fluid Condition by Means op Germ-free 

Filtration 15 

Preservation in the Froze* Condition 18 

Preservation in Dry Powder Form 19 

Preservation Dried on Paper 20 

Preservation by Mixing with Sodium Sulphate 21 

Preservation Within the Living Animal. ,,,,,, 21 

Diagnostic Methods 
Precipitin Reactions 

Differentiation between Human and Animal Blood or 
Flesh 22 

Recognition of Meat Adulteration — Method of Uhl- 
enhuth, Wiedanz and Wedermann 28 

Serum Diagnosis of Syphilis: Method of Fornet and 
schereschewsky , 35 

Floccule Forming Reactions 

Serum Diagnosis of Syphilis: Method of Porges and 

Meier 37 

Method of Elias, Neubauer, Porges and Salomon ... 38 

Method of Klausner 39 

The Agglutination Reactions 

Identification of Unknown Bacteria Recovered from Water, 
Dejecta, etc. 

Macroscopic Method of Proscher 42 

Microscopic Method (Hanging Drop Method) 45 

Testing Patient's Blood for Agglutination (Widal Test) 

Macroscopic Method of Proscher 46 

Macroscopic Method of Kafka 50 

Microscopic Method (Hanging Drop Method) 52 

Diagnosis of Mixed Infection (Saturation Method of 

Castellani) 53 



CONTENTS. 



Bactericidal Reactions 
In the Living Animal (Pfeiffer's Phenomenon) 
Identification of Unknown Bacteria Recovered from 



Water, Dejecta, etc 56 

Demonstration of Antibodies in Patient's Serum 59 

In Vitro 

Demonstration of Bactericidal Substances in Patient's 
Serum 60 

Cytolytic Reactions 

Cancer Diagnosis of Freund and Kaminer 64 

Diagnosis of Pregnancy (Improved Method of Abder- 

halden) 68 

"Clinical Alexin Test" of Moro 72 

Antihamolytic Reactions {Complement Deviation Tests) 

Demonstration of Proteid Antigens, (e.g., of Human Pro- 
teid in Blood Stains) 75 



Determination of Antibodies in Serum, Cerebrospinal Fluid 
or Tissues of Patients 
Syphilis Diagnosis 
Method of Wassermann and Meier (Berlin Method). . . 85 



Method of Taege (Breslau Method) 89 

Method of Hoehne (Frankfurt Method) 93 

Simplified and Improved Method of Stern (Breslau 

Method) 94 

Simplified Method of Bauer 96 

Simplified Method of Tschernoguboff 98 

Method of Landsteiner, Potzl and Muller 100 

Cancer Diagnosis 

Method of von Dungern 101 

Gonorrhoea Diagnosis 

Method of Schwartz and McNeil 104 



xii CONTENTS. 

Tuberculosis Diagnosis 
Method of Hammer 107 

Cobra Venom Reactions 

Inhibition op Cobra Venom Hemolysis, Psychoreaction 
of Much and Holzmann 109 

Increased Resistance to Cobra Venom Hemolysis (Syph- 
ilis Diagnosis, Method of Weil) Ill 

Activation of Hemolysis of Horse's Blood-cells in Cancer 
(Method of Kraus, Graff, Ranzi) , 113 

Antifermentative Reactions 

Estimation of Antitrypsin Content of Blood 

Marcus' Modification of the Method of Muller and 

jochmann 115 

Method of Bergmann and Meyer 117 

Meiostagmin Reaction 
Method of Ascoli and Izar 119 

Phagocytic Reactions 

Estimation of Opsonin Content of Serum 

Method of Bine and Lissner 128 

Method of Kammerer 136 

Demonstration of Antistaphylolysin in Serum 137 

Determination of Diphtheria Toxin 142 



ILLUSTRATIONS 

FIG. PAGE 

1. Berkfeldt Filter 14 ^ 

2. Filter for Preservation of Serum in Fluid Form 16 ^ 

3. Uhlenhuth's Apparatus for Preventing Contamination 17 

4. Test Tube Rack 25 / 

5. Capillary Pipettes 33 ^ 

6. Tube for Obtaining Serum of Patient 129 • 

7. Capillary Tubes for Measuring Equal Volumes 132 V 



Muller's 
Serodiagnostic Methods 



Methods of Injecting Animals. 

Intraperitoneal Injection of Rabbits to produce 
Immune Serum (Method of Uhlenhuth). — An 

assistant grasps the rabbit's hind legs with his 
left hand, and the forelegs with his right, and 
holds the head down. Thus the intestines fall as 
far as possible into the upper portion of the 
abdominal cavity. The belly wall is turned to- 
ward the operator, who removes the hair from 
the lower part of the belly with scissors, cuts 
through the skin, which is raised for the purpose, 
with scissors sterilized in the flame and cooled, 
exposing a small area of the underlying muscle. 
The muscular coat is then carefully punctured 
with a dull trochar, the syringe adjusted, and the 
desired fluid injected. After the withdrawal of 
the trochar, the small wound is closed with cotton 
and collodion. If no assistant is available, the 
animal may be fastened to one of the usual animal 
holders. 

Intravenous Injection of Rabbits. — Long-eared 
animals are best adapted for injections into the 

l 



2 SERODIAGNOSTIC METHODS. 



marginal vein of the ear. Short-eared animals 
often occasion difficulty, especially to beginners. 
First remove the hair from the site of the injec- 
tion with scissors, then rub the entire ear briskly 
with a bit of cotton wet with ether, or the ear 
may be covered with cotton soaked in hot water, 
until the ear vessels become distinctly distended 
with blood. It is still better to stroke the ear 
from the base outward with an electrode during 
the entire procedure. Insert the needle, attached 
to the syringe, superficially alongside the vein, 
and carry it forward (toward the heart) for a 
short distance, to make sure that the point is 
really within the vessel lumen, and then inject 
slowly, under very slight pressure. Care should 
of course be taken to see that no air is injected. 
It is a good plan to place a spring hsemostat over 
the needle during the injection. Being held by 
the needle, it does not, of course, compress the 
vessel until the needle is withdrawn, when it at 
once compresses the vessel, preventing the escape 
of the injected material, as well as of blood. 



Methods of Deawing Blood. 

Drawing Blood from the Marginal Vein of the 
Rabbit's Ear to Obtain Small Amounts of Serum. — 

For this purpose also, long-eared rabbits are best 
adapted, since it is often impossible to obtain from 
short-eared animals more than a very small 



SEKODIAGNOSTIC METHODS. 3 



amount of serum. The vein is first dilated as des- 
scribed on p. 2, and is then incised with scis- 
sors or a sharp scalpel, as close as possible to the 
rabbit's head. As the blood drops from the wound, 
it may be caught either in a small test-tube, or, 
if only a fraction of a cubic centimetre is required, 
in a capillary tube (v. a, p. 7). The maximum 
amount obtainable by this method is about 10 c.c, 
or as much more, under favorable circumstances. 
To stop the bleeding, a bit of cotton may be laid 
on the wound and held in position with a spring 
forceps, which usually can be removed in a few 
minutes. A great advantage of this method lies 
in the fact that the same animal may be used 
repeatedly, in this way, to obtain serum. 

To separate the serum from the blood proceed 
as follows : If the blood has been received in capil- 
lary tubes, these are treated as described on 
p. 9. If, on the contrary, test tubes were used, 
the latter may be either : 

(a) Set aside for 24 hours at from 10 to 14° C. 
(e.g., placed in running water), when the serum 
which separates from the clot is transferred to a 
second tube; or, 

(b) After coagulation, the clot may be liber- 
ated from the sides of the tube with a platinum 
wire, the whole strongly centrifugated, and the 
serum thus separated immediately. 

As blood-cells easily become mixed with the 
serum when the latter is poured from a tube, it is 



4 SERODIAGNOSTIC METHODS. 



often necessary to centrifugate the serum a sec- 
ond time, and then carefully decant the serum 
from the sediment. The serum may be removed 
after the first centrifugation by means of a pipette, 
the point of which is bent upward. 

Drawing Blood from a Rabbit to Obtain Large 
Amounts of Serum. — (a) The animal is deeply 
chloroformed and placed in an animal holder ; the 
breast and abdomen are then rubbed with alcohol, 
to lessen the contamination of the blood by hair ; 
the soft parts are cut from both sides of the breast 
wall by a longitudinal incision, and the anterior 
breast wall is removed. Before the heart stops 
completely, a free incision is made into the heart 
chambers, and the animal is allowed to bleed to 
death into the pleural cavity, the lungs in the 
meantime having been removed for this purpose. 
The blood is transferred from the pleural cavity 
to a sterile graduate cylinder with a pipette hav- 
ing a broad lower opening. The blood may be 
left in the cylinder for 24 hours, at 10-15° C, 
or may be placed at once in test tubes, and, after 
coagulation, centrifugated. The further treat- 
ment is described above. 

(b) The following method may also be em- 
ployed: The animal being fastened down or held 
by an assistant, the operator grasps a large longi- 
tudinal fold of skin on the neck, and cuts it off 
with a single strong stroke of the scissors. The 
neck muscles, and below these the trachea, are 



SERODIAGNOSTIC METHODS. 



5 



next exposed for a considerable distance. With 
forceps and scissors, the muscles are dissected 
away, and the carotid is exposed. This having 
been separated by dull dissection, two small spring 
haemostats are placed on it close together, and 
the vessel is severed between them. The proxi- 
mal end is then held with the forceps within the 
mouth of a sterile, wide-mouthed vessel. The 
wall of the carotid is now incised with fine scissors 
proximal to the forceps, and the blood is allowed 
to flow into the vessel. The yield of blood may 
be somewhat increased by pressing on the ani- 
mal's abdomen and thorax. The further treat- 
ment is as given on p. 4. 

(c) Finally, a third method of obtaining blood, 
that of Heim, is as follows : 

To obtain blood which is certainly sterile, pre- 
pare a glass tube about 25 cm. long by 16 mm. di- 
ameter, bent in an obtuse angle and drawn out 
several centimetres at its lower end, which is 
finally drawn out to a capillary point. The 
upper end is plugged with cotton, and the whole 
sterilized. After the animal has been fastened 
to the holder, the carotid is exposed, clamped 
with a small artery clamp, and a ligature is 
passed around it proximal to the clamp. The 
capillary end of the tube above described is 
then broken off, and the end is inserted into 
the artery, pointing toward the heart. Care 
should of course be taken to see that the tube is 



6 SERODIAGNOSTIC METHODS. 



not thrust completely through the artery and out 
again on the opposite side. The ligature is then 
tied by an assistant, so as to include the tube. 
Under favorable conditions the blood will flow 
readily into the tube. Any clots which may form 
may be removed by careful suction over the cot- 
ton plug. About 60 c.c. may be obtained in this 
manner from a large animal. If a less amount 
is desired, the tube is removed earlier, the artery 
being ligated, first on the peripheral and then on 
the central side. 

The tube filled with blood may be sealed at its 
lower end with sealing wax and then centrif ugated 
in order to separate the serum, or it may be left 
in a cool place for 24 hours. 

Methods of Obtaining Blood from Other Ani- 
mals — (a) The most convenient method of ob- 
taining blood from larger animals, such as the 
horse and goat, is to distend the jugular vein by 
means of a rope drawn about the neck, and then to 
open the vein with a sterile trochar, first having 
shaved and thoroughly disinfected the overlying 
skin. (In the goat, the large vein on the inner 
surface of the hind leg may be reached even more 
easily. — Translator.) 

(b) Sterile blood may also be obtained easily 
from a newly slaughtered animal. The first flow of 
blood is discarded, and the remainder is collected, 
under strict asepsis, in sterile glass cylinders of 
about 600 c.c. capacity and 6 cm. diameter. After 



SERODIAGNOSTIC METHODS. 7 



the clot separates the serum is removed with 
sterile pipettes of 50 c.c. capacity, and is pre- 
served in the ice box, in rather wide test tubes. 
A more complete separation of the serum may be 
secured by placing a sterile weight on the clot 
during the coagulation. If there is any danger of 
contamination, the serum must be filtered through 
a sterile Berkefeldt filter. 

Chicken 's blood may be obtained most easily 
from the wing vessel, lying on the inner aspect of 
the wing ; or it may be obtained from the carotid, 
care being taken to avoid the oesophagus and 
trachea. 

Obtaining Small Amounts of Human Blood- 
Serum. 

For obtaining small amounts of blood-serum, 
such as is required for the Widal reaction and 
most other serum reactions, the following method 
is recommended: 

Method of Proscher. 1 — Apparatus. — (a) IT- 
shaped, or simple straight tubes of thin glass, of 
about 2 mm. internal diameter, and drawn out to 
a point at one end. The length of the tube or 
"capsule" is regulated by the size of the centri- 
fuge to be used later. 

(b) Alcohol-ether and cotton. 



1 Zentralbl. f. Bak., etc., vol. 31 (1902). 



8 



SEKODIAGNOSTIC METHODS. 



(c) A sharp scalpel, preferably double-edged, 
or a lancet with a platinum-indium point, actu- 
ated by a spring. 

(d) Sealing wax or some other material suit- 
able for sealing. 

(e) A file or a glass cutting knife. 

(/) Chloride of iron, for use as a styptic, will 
be found convenient. 

Technique. — First clean the lobe of the ear with 
alcohol-ether. An incision about 1 cm. long and 
fairly deep is then made on the outer border, near 
the tip. The blood should not be allowed to drop 
off, this being prevented by sponging with a dry 
clean cloth. Apply one of the tubes above de- 
scribed to the drop of blood, or lay it directly in 
the incision. The blood is drawn into the tube 
by capillarity. If one tube becomes plugged, an- 
other is quickly substituted. With patience and 
some practice a sufficient quantity of blood can 
always be obtained. If the bleeding is too free, 
it can always be checked easily by applying a 
sponge wet with chloride of iron. The desired 
quantity of blood can also be obtained easily from 
the finger-tip, by using a tourniquet, or by first 
swinging the arm vigorously for a moment. After 
filling, the tubes are sealed at each end with wax. 
It must be remembered, however, that if the glass 
is wet, the wax will not stick to it. If this is the 
case, the outside of the tube may be warmed care- 
fully till dry. The sealed tubes can be mailed if 



SERODIAGNOSTIC METHODS. 9 



necessary. The serum is finally separated from 
the clot by placing the tubes in centrifuge tubes, 
the bottom of which is covered with cotton, and 
centrifugating. The waxed ends are then cut off 
with the glass cutter, and a mark is made with 
the file at the boundary line between the serum 
and the clot. The tubes are broken at this point, 
the serum being prevented from escaping by capil- 
lary attraction. Should the clot, as may happen, 
cling so tightly to the wall of the tube that the 
serum fails to separate, it should be loosened with 
a slender platinum wire, and the centrifugation 
repeated. The tube may then be opened at the 
boundary line as above described. The serum is 
then drawn directly from the tube into the meas- 
uring pipette, by applying the former, held hori- 
zontally, to the point of the latter, held vertically. 
In this manner any loss of serum is prevented, 
again by capillarity. Care must be taken to see 
that there are no bubbles of air in the mouth of 
the tube, which might pass over into the pipette ; 
such bubbles are easily removed by touching the 
end of the tube with filter paper. 

Method of Czaplewski-Schottelius. 2 — Instead 
of the customary thin-walled glass tubes, 
Czaplewski uses small, thick-walled centrifuge 
tubes, with a pointed lower end (length 5.5 cm., 
diameter above 0.8 cm., below 0.4 cm.). The 



2 Miinch. med. Woch. (1906), p. 508. 



10 SERODIAGNOSTIC METHODS. 



tube is provided with a tightly fitting cork, 
through which passes a long, stout insect needle. 
The point of the needle is bent in a hook, and cov- 
ered with the best fat-free, purified absorbent 
cotton. A small portion of the cotton extends 
beyond the point of the needle. (These tubes 
can be obtained from Theo. Schroter, Leipsic- 
Connewitz.) 

Thoroughly soak the cotton with the blood, as 
it issues from the incision. The cork is then re- 
placed, and the tube may be mailed, if desired. 
Centrifugate to obtain the serum. A surpris- 
ingly large yield may be obtained. The serum is 
removed from the tube with a pipette. This 
method is simpler and calls for less skill than 
Proscher's method. The only equipment needed, 
beside the tubes above described, is a scalpel, 
tincture of iodine, cotton, and chloride of iron. 

Obtaining Larger Quantities of Human Blood. 

Larger quantities, according to Uhlenhuth, 
can be most easily obtained in one of the following- 
ways : 

Placental Blood — As soon as the child's head 
presents, sterile towels are spread under the 
mother from the buttocks to the knees. The 
attendant, wearing sterilized rubber gloves, places 
the infant on these towels. The cord is then sev- 
ered, and the placental end is compressed. Care- 
fully insert the end of the cord in the mouth of a 



SERODIAGNOSTIC METHODS. 11 



large, sterile, glass flask or cylinder, after flaming 
the latter 's lip. Then, by pressure on the uterus, 
squeeze out as much of the blood present in the 
placenta as possible. The flask is then closed with 
a sterile cotton plug. 20 to 30 c.c. may be obtained 
from each placenta in this way. 

The use of the Heurteloup cupping appa- 
ratus, such as is employed by ophthalmologists, is 
also recommended. This yields about 15 c.c. of 
fluid blood. 

Veni-Puncture or Phlebotomy. — The upper 
arm of the patient is compressed with a rubber 
bandage, so as to distend the veins. Eub the skin 
vigorously with a bit of cotton wet in alcohol, and 
pass a hypodermic needle into the vein at the 
bend of the elbow. As the blood drops from the 
needle it is received in a broad test tube. (The 
rubber bandage should always be removed before 
the needle is withdrawn from the vein. — Trans- 
lator.) After the needle is withdrawn the opening 
is closed with adhesive plaster. 

Finally, the blood of cadavers can be used, 
according to Ziemke, provided it can be obtained 
sterile. 

Method of Obtaining Defibrinated Blood and 
Red Blood-Cells.— For this purpose, those methods 
of drawing the blood, in which it flows rapidly and 
in a full stream are preferable. The blood is re- 
ceived in thick-walled glass vessels, containing 
glass or porcelain beads (these are apt to break 



12 



SERODIAGNOSTIC METHODS. 



and become pulverized. — Translator) , or steel 
shavings, or steel ball bearings or bits of glass 
rod. The vessels used may be bulbs, cylinders, 
salt bottles, etc. One should be careful to see 
that they are not filled too full. They should be 
closed with wood or rubber stoppers, not with 
cotton. After the blood is placed in the bottle, the 
latter is shaken vigorously for at least five 
minutes. 

To free the blood-cells of serum, the defibrin- 
ated blood is diluted with nine parts of 0.86 per 
cent, sodium chloride solution, and centrifugated. 
The clear supernatant fluid is then poured off, 
an equal amount of fresh salt solution is added 
(mixed thoroughly. — Translator), and the centrif- 
ugation is repeated. This process, called "wash- 
ing the red blood-cells," is repeated at least three 
times. 

The red blood-cells may also be cleared of 
serum without defibrinating, by receiving the 
blood in some fluid which prevents coagulation. 
For this purpose one may use either, (a) a 1.5 per 
cent, solution of sodium citrate, or (b) an 0.86 
per cent, solution of sodium chloride, to which 
0.1 per cent, to 0.5 per cent, of potassium oxalate 
has been added. 

Inasmuch as many sera (especially those de- 
rived from the sick or from immunized animals) 
coagulate with extraordinary rapidity, it is advis- 
able to bring the blood as quickly as possible in 



SEEODIAGNOSTIC METHODS. 13 



contact with the greatest possible amount of these 
coagulation-preventing fluids. Thus, for example, 
nine parts of the fluid may be placed in a gradu- 
ated cylinder, the blood allowed to flow in to the 
mark 10, and the contents thoroughly mixed at 
once. By centrifugating and washing three times 
with the usual 0.86 per cent, sodium chloride solu- 
tion, the serum or plasma is removed at the same 
time with the coagulation-preventing substance. 

The Peeseevation of Seea. 

Sera of Normal Animals — Normal sera, des- 
tined to be used for purposes of immunization, 
are best kept in well-corked medicine bottles hold- 
ing about 100-200 c.c. One may add 0.5 per cent, 
carbolic acid (i.e., 1-10 volume of 5 per cent, car- 
bolic acid solution) or 1 per cent, chloroform or 
toluol as a preservative. If either of the last two 
is used the serum should be shaken vigorously 
from time to time to aid the solution of the anti- 
septic in the serum. At the point of contact be- 
tween the serum and the chloroform or toluol a 
whitish cloud forms, consisting of precipitated 
albumin. This usually does not extend further. 
The bottle should be kept in a cool, dark place. 

Sera to which chloroform or toluol has been 
added may be placed in an open shallow dish in 
the incubator, for a short time before being in- 
jected, in order to remove the preservative. 



14 SERODIAGNOSTIC METHODS. 



Eecently a preservative has been recommended 
consisting of a mixture of 2 c.c. of formalin with 
100 c.c. of 0.9 per cent. NaCl solution. This is 
mixed with the serum in a proportion of 1 : 4, 1:3, 
or 2 : 3. 3 



Fig. 1. 




IMMUNE SEEA. 

Immune sera may be preserved either in the 
fluid or in the dry form. 

Preservation in Fluid Form by Means of Anti- 
septics — Agglutinating sera retain their activity 

3 Leers, Methoden und Tecknik der Gewinnung, Priifung, und 
Konservierung des zur Eiwessdifferenzierung dienenden Anti- 
serums, Berlin, 1908, R. Schoetz. 



SERODIAGNOSTIC METHODS. 15 



practically unchanged in the presence of from 
0.3 per cent, to 0.5 per cent, carbolic acid solution. 
Therefore they are usually preserved in this man- 
ner. Precipitating sera may be preserved in the 
same way, or by the addition of a small amount 
of toluol (1 per cent.). According to Uhlenhuth, 
Weidanz and Wedemann 4 it is still better to filter 
such sera through a sterile Berkefeldt filter (see 
Fig. 1) and to store them without any antiseptic. 

Preservation in Fluid Form by Germ-Free Fil- 
tration. — In a negative pressure flask (Fig. 2, b) 
connected by a rubber cork with a Berkfeldt filter 
(a) is placed a sterile test tube, into which the 
serum drops after filtration. The long side arm 
of the filtration flask is connected with a water 
pump (d) by a tube in which is a glass valve (e) 
and a back pressure valve (c). When the serum 
has all passed through, the rubber stopper is re- 
moved, the test tube lifted out with sterile forceps, 
and the end closed with a scorched cotton plug. 
The test tube is allowed to stand for some days, 
at room temperature, to make certain whether the 
filtration needs to be repeated. If the serum 
proves to be sterile, it is transferred with a sterile 
pipette into small brown vials of 1 c.c, capacity. 
These vials are closed temporarily with cotton 
only and allowed to remain for some days at room 
temperature. They are finally carefully sealed 



4 Arb. aus dem Kaiserl. Ges.-Amt., vol. 28 (1008). 



16 SERODIAGNOSTIC METHODS. 



hermetically, but not until one is convinced tliat 
they are sterile by the fact that the contents 
remain clear. 



Fig, 2. 



SERODIAGNOSTIC METHODS. 17 



Uhlenhuth and his collaborators have designed 
a special apparatus for handling the fluid, in order 
to prevent with certainty contamination of the 
serum, which may easily occur after this sort of 
filtration. The apparatus is a combination of a 
bacterial filter and a serum filling-pipette 7 so 



Fig. 3. 




arranged as to carry the filtered serum directly 
into the small brown vial. The apparatus may be 
obtained from the Kny-Scherer Co., New York 
(Fig. 2). Uhlenhuth and Weidanz have designed 
a special apparatus of small size, "Mikrofiltrier- 
abfiilapparat" (Fig. 3), for the filtration of very 
small quantities. Detailed directions for the use 
of both pieces of apparatus are given in the article 

2 



18 SERODIAGNOSTIC METHODS. 



by Uhlenhuth, Weidanz, and Wedemann, already 
cited. 

The imnmne serum thus obtained is best kept 
in the ice box. It should at least be protected 
from light and heat. In case the serum is stirred 
just before use, the fine precipitate which often 
develops after long standing, may be removed by 
centrifugating or by filtering again. Immune sera 
thus preserved retain their activity for months 
and years. 

Preservation in Frozen Condition -In any 

case in which the usefulness of the serum does 
not depend upon its remaining absolutely clear, it 
may also be preserved in a frozen condition. An 
ice machine capable of maintaining 0° is found 
only in large and well-equipped laboratories. For 
smaller laboratories the smaller freezing appa- 
ratus devised by Morgenroth and manufactured 
by F. & M. Lautenschlager, in Berlin, may be 
adapted to meet this requirement. It is called 
Frigo. A home-made freezing apparatus may 
be made as follows : A large kettle, holding several 
litres, is placed in a large wooden chest ; the space 
surrounding it tightly packed with sawdust, and 
a sheet-metal covered box is placed within the 
kettle, to hold a small serum flask. The kettle is 
then filled with ice or snow, around the metal box, 
so that a freezing mixture is formed which will 
last for some days, if the chest is covered over 



SERODIAGNOSTIC METHODS. 19 



with several layers of felt ; but it must be renewed 
every few days. 

Freezing the serum often renders it cloudy or 
causes precipitates. 

Preservation in Dry Form. — The serum may 
be spread out in the thinnest possible layer on 
large glass plates and dried in the incubator. 
After a few hours the dried serum, which adheres 
only slightly to the glass, can be removed with a 
spatula and stored in hermetically sealed tubes 
in vacuo. 

It is better to carry out the drying process also 
in vacuo. The large serum institutes employ a 
specially made serum drying apparatus; in the 
smaller institutes, however, the ordinary vacuum 
apparatus may be adapted to this purpose. One 
may prepare the simplest form of drying appa- 
ratus according to the following method of 
Taege : 5 A glass bell jar, with a ground base and a 
large opening at the top, is placed on a polished 
iron plate which can be heated with a gas burner 
or set in a water-bath. The opening at the top 
is closed with a three-hole rubber stopper; one 
hole carries a thermometer, a second is connected 
with a manometer, while the third carries a bent 
glass tube connected, by means of a thick walled 
rubber tube, with a water vacuum pump. 

The dried serum should be dissolved in physio- 
logical salt solution for use. 

5 Munch, med. Woch. (1908), No. 33. 



20 SBRODIAGNOSTIC METHODS. 



It is simpler and more convenient to dry 
on paper the amount of antiserum required for a 
single test, according to the method of Leers, 6 
since it is difficult to measure exactly the minute 
amounts of serum often needed for the applica- 
tion of biological reactions. The paper must be 
fairly firm in order to prevent drops soaking 
through. Black Natur papier (hat-box paper, — 
Translator) is convenient for this purpose. With 
a graduated pipette 0.1 c.c. of the serum is spread 
in a large drop on small pieces of paper; after 
remaining 2 to 4 hours in the incubator, the drop 
is dried to a firm, shining crust. This clings 
firmly to the paper, on which individual portions 
of the serum are preserved in an exsiccator pro- 
tected from light, heat, and moisture. Antiserum 
thus preserved retains its activity for a long time 
unaltered. 

When a test is to be made the bit of paper 
carrying the single portion of serum required is 
washed for a short time in sodium chloride solu- 
tion to remove dust and lint, and is then placed 
in a measured amount of salt solution; or it may 
be placed directly in the proteid solution to be 
tested, in such a manner that the paper side lies 
next to the solution. When the paper becomes 
soaked it sinks to the bottom, or it may be pushed 

6 Methoden und Tecknik der Gewiimung, Priifung, imd Kon- 
servierung des zur forensischen Blutdifferenzierung dienenden 
Antiserums, Berlin, 1908, R. Schoetz. 



SERODIAGNOSTIC METHODS. 



21 



down. One should, however, avoid forcing the 
paper under the surface of the fluid before it is 
soaked, as in this case the air is not completely 
removed from the meshes of the paper, and the 
solution of the serum is affected. 

The paper selected to dry the serum on should 
be one which does not give off much color to the 
solution within the time required to dissolve the 
serum. 

(Stokel 7 recommends mixing the serum with 
anhydrous sodium sulphate, 1 c.c. serum to 0.7-2.0 
grammes of the salt. The water of the serum is 
absorbed, leaving a solid which can be pulverized. 
By dissolving an aliquot portion (1.7-3 grammes) 
in 100 c.c. distilled water, an isotonic solution is 
obtained which represents a 1 per cent, solution 
of the serum. — Translator.) 

Preservation within the Living Animal — It is 
sometimes an advantage to remove only so much 
serum from the immunized animal as is needed 
for the test, without killing it. The animal may 
then be re-immunized from time to time, or it may 
be permitted to rest. Each time the animal is 
bled, the titre of the serum may be raised as de- 
sired by giving one or two injections. A disad- 
vantage of this method lies in the marked tend- 
ency shown by immunized animals to sicken, or 



'Wien. klin. Woch., 23 (1910), No. 43. 



22 SERODIAGNOSTIC METHODS. 



to die with symptoms of anaphylaxis or maras- 
mus. A larger number of animals must therefore 
be kept immunized, thereby adding materially to 
the expense. 

Technique of the Serodiagnostic Methods, 
precipitin reactions. 

Principle. — The mixing, in suitable propor- 
tions, of an immune serum of high potency with 
its homologous proteid antigen causes the for- 
mation of a specific precipitate. 

Practical Applications. — The identification of 
proteid substances of unknown origin, as, the 
differentiation of human and animal blood (ac- 
cording to Uhlenhuth), or the recognition of meat 
substitutions, etc.; the sero-diagnosis of syphilis. 

Differentiation of Human and Animal Blood 
or Flesh — Apparatus. — (a) A highly potent and 
absolutely crystal clear serum immune against the 
proteid to be recognized. It is best obtained by 
means of intravenous or intraperitoneal injection 
of a considerable number of rabbits with blood, 
or, still better, with serum. The injections should 
be repeated four or five times at intervals of five 
or six days. 

Not all the rabbits injected with a foreign 
blood or serum will furnish a sufficiently potent 
immune serum. From the third injection on, 



SERODIAGNOSTIC METHODS. 23 



frequent tests should be made*, with 2 or 3 c.c. of 
blood taken from the ear vein, to determine 
whether the rabbits' sera have attained a suffi- 
ciently high titre. If the serum tested shows the 
desired characteristics, the animal is killed and 
its serum collected. The serum must possess the 
following properties : 

It must be absolutely clear. This can be se- 
cured by filtering through a sterile Berkefeldt fil- 
ter. Further, it must not show a milky opales- 
cence by transmitted light. Such an opalescence 
depends upon the stage of digestion in the animal 
at the time it is killed. Since* it oan not be re- 
moved by filtration, as is possible with precipi- 
tates, an opalescent serum must in every case be 
discarded. Animals whose blood when removed 
for the purpose of making a test shows opales- 
cence should not be killed until the opalescence has 
disappeared. This usually occurs after several 
hours' fasting. 

Finally, the serum must be of high potency 
and must react promptly. In order to make cer- 
tain of this, an accurate titration of the serum 
is made before the animal is killed, and again 
after the final preparation of the serum. This 
titration is made in the same manner as when 
carrying out the test, as described later. For the 
present, therefore, only the quantities of reacting 
substances to be used for this special purpose 
are given : 



24 SERODIAGNOSTIC METHODS. 



Dilutions of 1-1000, 1-10,000, and 1-20,000, 
respectively, of the serum or blood against which 
the immune serum acts are first prepared with 
physiological salt solution (0.8 to 0.9 per cent.). 
To 2 c.c. of each of these dilutions add 0.1 c.c. of 
the immune serum, without shaking. In the 
1-1000 dilution a distinct clouding should appear 
at once, or, at the most, after one or two minutes ; 
and, after three to five minutes, the beginning of 
the reaction should be distinctly recognizable in 
the higher dilutions. The reaction should be fully 
complete after thirty minutes at the most, at room 
temperature. 

The immune serum may be preserved in 
sterile, hermetically sealed glass tubes, after fil- 
tering through a sterile Berkefeldt filter. The 
serum will be rendered free of bacteria in this 
way, provided the filter works properly. Care 
should be taken to avoid using cracked filters. 

A safer way is to preserve the serum, unfil- 
tered, by the use of 0.5 per cent, carbolic acid 
(1-10 volume of 5 per cent, carbolic acid solution) 
as a preservative. 

(b) A solution of the unknown substance. 
Physiological salt solution only should be used 
as a solvent and diluent for blood- stains, etc. The 
dilution should be about 1-1000. In the case of 
blood-spots this is not always easy to estimate. 
Uhlenhuth gives the following directions for 
obtaining the proper dilution: 



SERODIAGNOSTIC METHODS. 25 



1. The dilution should be almost completely 
colorless by transmitted light. 

2. It should give only a slight cloud on heating 
with a few drops of nitric acid. 

3. It must foam freely on shaking, in spite of 
the high dilution. 

This solution also must be crystal clear, for 
which purpose nitration may be employed. 




(e) Test-tubes, 10 cm. long by 0.9-1 cm. wide. 
These must be absolutely clean. 

(d) A test-tube rack. The rack for 12 tubes 
devised by Uhlenhuth is very convenient. This 
has the holes for the tubes bevelled, and permits 
the test-tubes to hang suspended in the holes. 
Since the small test-tubes are often of unequal 
diameter, it is a good plan to select in advance 
tubes as nearly as possible of the same size. To 
avoid mixing the tubes, the holes in the rack may 
be numbered. (See Fig. 4.) 



26 SERODIAGNOSTIC METHODS. 



(e) Physiological salt solution. 

(/) Volumetric pipettes, 0.1 c.c. graduated in 
1-100 's (the calibration extending to the point), 
and 1 c.c. and 10 c.c. in 1-10 's. 

(g) Dried blood-clots from various species of 
animal to serve as controls. Dilutions approxi- 
mating 1-1000 should be prepared of each of these 
in the same manner as the unknown substance. 

Technique. — The following mixtures are set up 
in a series of test-tubes : 

1. The test proper: 2 c.c. unknown solu- 
tion + 0.1 c.c. immune serum. 

2. Control : 2 c.c. salt solution + 0.1 c.c. im- 
mune serum. 

3. Control : 2 c.c. diluted blood of that species 
of animal whose blood is suspected to be present 
in the unknown solution + 0.1 c.c. immune serum. 

4. Control : 2 c.c. unknown solution alone. 

5. Control : 2 c.c. diluted blood-serum of a dif- 
ferent species of animal from that suspected to be 
present in the unknown solution+0.1 c.c. immune 
serum. 

When the test proper and control No. 3 result 
positively, and all the others negatively, the pres- 
ence of the blood or proteid of the species sus- 
pected in the unknown is established. If the re- 
sult is negative, the species to which the un- 
known specimen belongs has to be determined 
with the aid of new antisera prepared for each 



SERODIAGNOSTIC METHODS. 27 

species, and employed in the manner above 
described. 

No test is to be regarded as positive unless, as 
in the preliminary tests of the immune serum, the 
beginning of the reaction manifests itself within, 
at the most, one or two minutes after the addition 
of the immune serum, as a faint, misty cloud at 
the bottom of the test-tube; and within five min- 
utes, at room temperature, this must become a 
thick, woolly cloud. At the end of another ten 
minutes a definite precipitate must have formed. 

Any cloudiness which develops later than 
twenty minutes after the beginning of the reaction 
has no significance. It is to be noted, further, that 
the tubes must not be shaken during the perform- 
ance of the test. 

When only very limited amounts of the un- 
known substance are available, the test, accord- 
ing to Hauser, can be carried out in slender, 
scrupulously clean capillary tubes. A small 
amount of the unknown solution is drawn into the 
tube, and is then underlaid with a tiny amount of 
the immune serum — only so much, indeed, as will 
rise into the tube by capillarity. At the line of 
contact between the two fluids there will form a 
distinct grayish-white ring. 

If the reaction is carried out as above de- 
scribed, no heterologous reactions occur. The 
clouding, in other words, is very strictly specific, 
as Nutall has shown on the basis of 16,000 blood- 



28 SERODIAGNOSTIC METHODS. 



tests. Only as between very closely related spe- 
cies — horse and ass, sheep and goats, dog and 
fox — can any doubt possibly arise. Beef and 
sheep's blood are readily distinguished. 

The reaction can be obtained from blood in an 
advanced state of putrefaction, or from a clot 
which has been dried for a year. 

Inasmuch as proteids give the reaction, not 
only blood but other proteid-containing substances 
can be used for the test; for example, spermatic 
fluid, pus, purulent sputum, urine, etc. It is well 
to remember this in medico-legal practice. 

The application of the precipitin reaction to 
the detection of meat adulteration rests upon the 
same principle. 

Recognition of Meat Adulteration (Method of 
Uhlenhuth, Wiedanz, and Wedermann). 8 — Appa- 
ratus. — (a) A serum immune against that spe- 
cies of flesh which is to be determined in the un- 
known specimen. Usually it is merely a question 
of the recognition of horseflesh. The serum is ob- 
tained from rabbits by immunizing them against 
defibrinated horse blood, or, better, horse serum. 
The animals are injected, preferably intra- 
venously, every 5 days, with amounts varying 
from 2 to 3 c.c. Five or six days after the third 
injection, blood is drawn from the ear vein, and 
the serum which separates from the clot is 



Arbeiten a. d. Kaiserl. Ges. Amt, vol. 28 (1908). 



SERODIAGNOSTIC METHODS. 29 



titrated. The titration is carried out as follows: 
1 c.c. of a 1-1000 solution of horse serum, in 0.85 
per cent, sodium chloride solution, is mixed with 
0.1 c.c. of the perfectly clear and non-opalescent 
immune serum, without shaking. If a cloudiness 
develops at the bottom of the test-tube at once, or 
at the latest after one or two minutes, the anti- 
serum meets the requirements, and the animal 
may be killed and its serum separated. 

Out of a considerable number of animals thus 
immunized, only a small part will yield a usable 
serum after the third injection. Eabbits whose 
serum yields a distinct precipitate, but whose 
serum is too weak, are given further injections, 
preferably intraperitoneally or subcutaneously. 
Those which show no precipitin after the third 
injection may be given further injections intra- 
venously. Occasionally such animals furnish an 
active serum after the injection of from 60 to 
120 c.c. of serum, all told. 

The immune serum must fulfil the following 
requirements : 

1. It must be absolutely clear and sterile. This 
may be attained by filtering through a Berkefeldt 
filter. 

2. It must not be opalescent. To avoid this, 
the animal should be kept fasting for some hours 
before being killed. 

3. It must be highly potent. The titrating of 
the serum, to determine the degree of its potency, 



30 SERODIAGNOSTIC METHODS. 



is carried out exactly as described on page 
24. Its titre must reach 1-20,000; that is, 
it must show beginning cloudiness within five 
minutes, when mixed with horse serum diluted 
1-20,000. -Such antihorse serum may be obtained, 
until further notice, from the Kaiserlichen Ge- 
sundheitsamt, in Berlin. 

(b) An extract of the unknown meat. To pre- 
pare this, about 30 grammes of flesh is removed 
from the deeper parts of the specimen through a 
fresh opening. It should contain as little fat as 
possible, and should be removed with a knife 
which has been sterilized either by boiling or in 
the flame. It is then placed on clean writing 
paper, which in turn is placed on an absolutely 
clean, sterile tile, or other suitable base, and is 
ground up, preferably with a sterile chopping 
knife. After being ground it is placed in a sterile 
Erlenmeyer flask of about 100 c.c. capacity, in 
which it is broken up with a sterile glass rod, and 
is then covered with 50 c.c. of sterile 0.85 per cent, 
sodium chloride solution {not tap or distilled 
water!). Salted meat may be freshened first in 
a large sterile Erlenmeyer flask, in which it is 
covered with sterile, distilled water, renewed sev- 
eral times in the course of ten minutes, without 
shaking. 

The mixture of meat and salt solution should 
stand for 3 hours at room temperature, or over 
night in the ice box. Especially when the meat 



SERODIAGNOSTIC METHODS. 31 



contains a good deal of fat, a few drops of chloro- 
form should be added to facilitate solution. To 
determine whether a sufficient quantity of proteid 
substances has passed into solution, place about 
2 c.c. in a test-tube and shake vigorously. If a 
fine, bubbly foam develops, and persists for some 
time, the solution is ready for further treatment. 

The extract must then be filtered perfectly 
clear. When the meat used is lean and fresh 
this is readily accomplished by a single filtration 
through a hard filter paper (Schleicher and 
Schull, No. 575, 603, or 605), moistened with 
sodium chloride solution. When fat or salt meat 
is being examined, filtration through sterilized 
rotten stone is preferable. 

The latter is carried out as follows : The steril- 
ized rotten stone is worked up to a thin paste 
with sterile 0.85 per cent, sodium chloride solu- 
tion. This is then spread out in a layer about 
2 mm. thick on the filtering plate of a Buchner 
funnel, which has been previously carefully cov- 
ered with filter paper, in order to prevent the rot- 
ten stone from passing through the funnel. For 
the rest, the apparatus is arranged exactly as 
described on page 15. A clear filtrate can be 
very quickly obtained with the aid of a vacuum 
pump. In place of the rotten stone paste, one can, 
of course, use the much more expensive Berkefeldt 
rotten stone (Kieselguhr) bougie, which must, of 
course, be sterilized each time before use. 



32 SERODIAGNOSTIC METHODS. 



A clear filtrate having been obtained, this must 
then be further diluted, if it contains more than 
1 part of proteid in 300 parts of salt solution. To 
determine this, 1 c.c. of the filtrate is placed in a 
small test-tube and heated, and 1 drop of nitric 
acid, sp. g. 1.153, is added. If a marked cloud 
develops, which settles immediately to the bottom 
as a floceulent precipitate, it indicates that the 
solution is still too concentrated, and that it must 
be further diluted. At the point at which the 
addition of the nitric acid causes only a diffuse, 
opalescent cloudiness, which sinks to the bottom 
after five minutes as a barely perceptible precipi- 
tate, the desired concentration of the solution has 
been reached. Before proceeding with the test 
the reaction of the solution to litmus paper should 
be tested. It must be neutral, or only very slightly 
acid or alkaline. To avoid the possible addition 
of an excess of alkali, in case neutralization is 
necessary, it is convenient to use magnesium oxide 
rather than a soluble alkali. 

(c) As controls, extracts of pork and beef, 
prepared as above. 

(d) Perfectly clear normal rabbit serum. 

(e) Physiological salt solution. 

(/) Volumetric pipettes, 1 c.c. graduated in 
1/10 's, sterile. 

(g) Capillary pipettes. These can be made by 
drawing out glass tubing 5 mm. in diameter over 



SERODIAGNOSTIC METHODS. 33 



a blast lamp (Fig. 5). One calibrates these by 
measuring into a watch crystal 0.1 c.c. NaCl solu- 
tion from a slender, accurately calibrated pipette 
and then sucking this up into the capillary pipette. 
Allow the solution to drop slowly from the pipette, 
counting the drops. The number of drops ap- 
proximately equals 0.1 c.c. 

Fig. 5. 



(h) Test-tubes about 10 cm. long by 0.9 to 
1 cm. wide. 

(i) Test-tube stand. 

If the antiserum has been preserved in small, 
hermetically sealed tubes, and if one is not cer- 
tain that the contents of the other tubes came 
from the same rabbit, the contents of a single tube 
3 



34 SERODIAGNOSTIC METHODS. 



only may be used for a test, since precipitates 
sometimes occur from mixing antisera derived 
from different animals. This might lead to mis- 
takes in the interpretation of the result. 

If, on long standing, a precipitate has devel- 
oped in an antiserum, this should not be stirred 
up, but the clear, supernatant serum should be 
carefully drawn off with a capillary pipette. 

In handling the different solutions fresh, 
sterile pipettes should always be used. The rab- 
bit serum should be added by means of a capillary 
pipette carefully, and in such a manner that it 
flows down the side of the tube and collects at the 
bottom. The tubes should be allowed to stand at 
room temperature and must not be shaken. 

Technique. — The following mixtures are set 
up: 

Tube 1, 1 e.c. unknown extract + 0.1 c.c. antihorse serum. 
Tube 2, 1 c.c. unknown extract + 0.1 c.c. normal rabbit serum. 
Tube 3, 1 c.c. horse flesh extract + 0.1 c.c. antihorse serum. 
Tube 4, 1 c.c. pork extract + 0.1 c.c. antihorse serum. 
Tube 5, 1 c.c. beef extract + 0.1 c.c. antihorse serum. 
Tube 6, 1 c.c. sterile salt solution + 0.1 c.c. antihorse serum. 

If a misty cloudiness appears in tubes 1 and 3 
within five minutes, becoming denser within an- 
other five minutes, and settling within thirty min- 
utes to the bottom as a precipitate, the other tubes 
remaining clear, horseflesh, or the flesh of some 
other single-toed animal is present. (Cf., p. 28.) 
Cloudiness which dpvelops later than the above 



SERODIAGNOSTIC METHODS. 35 



should not be regarded as indicating a positive 
reaction. The earliest appearance of the cloud 
can be recognized more easily if the tubes are 
looked at by reflected or artificial light, with black 
paper or some similar substance placed behind the 
test-tube. 

Lues Diagnosis (Method of Fornet and 
Schereschewsky). 9 — Principle, — The sera of ta- 
betics and general paralytics give a positive pre- 
cipitin reaction with the serum of syphilitics. 

Apparatus. — (a) The perfectly clear serum of 
a tabetic or general paralytic. It is best to obtain 
the blood in the early morning, before breakfast. 
It is a good plan to filter it through Schleicher 
and Schull paper No. 602. 

(b) Serum of the patient to be tested, similarly 
obtained and treated. The sera may be preserved 
by the addition of formalin, 1-10,000, or by sealing 
in thin, sterile glass tubes. 

(c) As controls, the serum of a known syphi- 
litic and of a known non-syphilitic individual. 

(d) Test-tubes, 8 cm. long by 0.5 cm. wide. It 
is convenient to set these in a black rack, to the 
back of which a piece of black cloth is attached. 

(e) Pipettes, 1 c.c. in 1/100 's. 

Technique. — Dilutions of the sera (a) and(5), 



9 Deutsch. med. Woch. (1907), No. 41; Munch, med. Woch. 
(1907), No. 30. 



36 SERODIAGNOSTIC METHODS. 



and of the control sera (c) are first made in the 
proportions 1-5 and 1-10. Under- or over-lay 
0.15 c.c. to 3.0 e.c. of the diluted serum (a) with the 
diluted serum (b). The specifically heavier fluid 
(less highly diluted) is placed first in the test-tube 
with a pipette, and the lighter fluid is then allowed 
to flow from the pipette into the tube held on a 
slant. 

In this manner are prepared the following 
combinations, the figures in parentheses referring 
to the degree of dilution : 



a + 5 

a + & (5x) 
a + & (lOx) 



a (ox) + & 

a (5x) + & (5x) 

a (ox) + 6 (10x) 



a (lOx) + & 

a (lOx) + 6 (5x) 

a (lOx) + 6 (lOx) 



In the same manner the controls (c) are under- 
or over-laid with the dilutions of {a). Tests in 
which the layering is not perfect must be 
discarded. 

After 2 hours, at the most, at room tempera- 
ture, a delicate ring appears in positive reactions, 
at the point of contact of the fluids, which becomes 
especially distinct if one reflects the daylight fall- 
ing on the tubes by means of a piece of black paper 
held obliquely behind the tubes. The controls made 
with a non-luetic serum must remain negative, 
while those made with the known luetic serum 
must be positive, if the reaction is to have any 
significance. 



SERODIAGNOSTIC METHODS. 37 



Floccule Fokming Eeactions. 

Principle. — In certain diseases, notably syph- 
ilis, the blood-serum develops a flocculent cloudi- 
ness when mixed with colloidal solutions of leci- 
thin or sodium glycocholate. 

Practical Applications. 
Lues Diagnosis (Method of Porges and 
Meier). 10 — Apparatus. — (a) 1 or 2 c.c. of the 
patient's blood-serum, diluted 1-5 with physio- 
logical salt solution; and similar solutions of 
knoivn syphilitic and known non-syphilitic blood- 
sera, to serve as controls. 

(b) Lecithin (Kahlbaum), rubbed up with 100 
parts of physiological salt solution to a 1 per cent, 
suspension, and shaken till homogeneous. 0.5 per 
cent, carbolic solution may be added as a preserva- 
tive. The suspension remains unaltered for 
several weeks. 

(c) Narrow test-tubes, 5 to 7 mm. in diameter. 

(d) Volumetric pipettes, 1 c.c. in 1-10 's. 
Technique. — 1 c.c. diluted serum is mixed with 

0.2 c.c. of the lecithin suspension and placed in the 
incubator for 1 hour. The result of the reaction 
is a floccule formation, developing diffusely 
throughout the whole column of fluid, at first very 
fine, but becoming steadily coarser. With strongly 
reacting sera, an actual precipitate forms, leaving 



10 Berl. klin. Woch. (1908), No. 15. 



38 



SERODIAGNOSTIC METHODS. 



the supernatant fluid more or less clear. The 
reaction may begin at once, but, as a rule, it is 
recognizable only after from % to 24 hours. The 
use of a lens for the observation of the reaction 
is, in general, to be avoided. The controls set up 
with normal serum must, of course, remain per- 
fectly clear. The method appears to give prac- 
tically the same results as the complement 
deviation test. 

Lues Diagnosis (Method of Elias, Neubauer, 
Porges and Salomon). 11 — Apparatus, — (a) The pa- 
tient's serum inactivated. About 0.5 c.c. is re- 
quired. It must be perfectly clear. As controls, 
known syphilitic and known non-syphilitic sera. 

(b) Freshly prepared 1 per cent, solution of 
sodium glycocholate (Merck). 

(c) Small test-tubes, 6 or 7 mm. in diameter. 

(d) Volumetric pipettes, 1 c.c. in 1-10 's. 
Technique. — 0.2 c.c. each of serum and of the 

solution of sodium glycocholate are mixed and left 
to stand for 16 to 20 hours at room temperature. 
They should be disturbed as little as possible. 
In positive cases floccules form by the end of this 
period, which usually collect in the upper layers 
of the fluid. Cloudiness or mere traces of floccule 
formation is to be regarded as negative. The 
following modifications of the test are not 
permissible : 



"Berl. klin. Wocli. (1908), No. 23. 



SERODIAGNOSTIC METHODS. 39 



1. Layering of the two reacting substances. 
A ring-like cloud always develops in this case. 

2. The use of the incubator, on account of bac- 
terial growth. 

3. The use of a solution not freshly prepared. 

4. The addition of carbolic acid to the solution 
of bile salts. 

5. The use of sera which are cloudy, non-homo- 
geneous, or which contain considerable haemo- 
globin. 

6. The use of special aids in judging the reac- 
tion, such as a lens or lateral illumination. The 
reaction is to be regarded as positive only when 
the floccule formation is plainly visible macro- 
scopically. The method seems to give practically 
the same results as the complement deviation test. 

Lues Diagnosis (Method of Klausner). 12 — Ap- 
paratus. — (a) About 1 c.c. of the patient's blood- 
serum. 

(b) Small test-tubes, 5 to 7 mm. in diameter. 

(c) Volumetric pipettes, 1 c.c. in 1-10 's. 

(d) Distilled water. 

Technique. — 0.2 c.c. of the serum and of the 
5 and 10 fold dilutions of the same are mixed with 
0.7 c.c. of distilled water, thoroughly mixed and 
allowed to stand at room temperature for from 
2 to, at the most, 15 hours. At the end of this 
time, if the serum is derived from a luetic patient, 



12 Wien. klin. Woch (1908), No. 7. 



40 SERODIAGNOSTIC METHODS. 



there will be found at the bottom of the test-tube 
a precipitate of globulin, 2 to 4 mm. deep, which 
also often floats in part in the fluid, forming 
plainly visible floccules. The reaction seems to be 
non-specific. 

Lues Diagnosis (Method of Hermann and 
Perutz). 13 — Apparatus.— (a) About 1 c.c. of the 
patient's blood serum, inactivated by heating for 



one-half hour at 56°. 

(b) The following solutions : 

(1) Sodium glycocholate 2.00 

Cholesterin 0.40 

Alcohol, 95 per cent 100.00 

For use, this is diluted 1-20 with distilled 
water. 

(2) Sodium glycocholate 2.00 

Distilled water 100.00 



This will not dissolve completely, but forms a 
suspension which is to be thoroughly shaken be- 
fore use. 

(c) Small test-tubes, 5 to 7 mm. in diameter. 

(d) Volumetric pipettes, 1 c.c. in 1-10 's. 

(e) An incubator at 21° is convenient, but not 
necessary. 

Technique. — Mix together one part of solution 
1, diluted as described, 1 part of solution 2, and 2 
parts of the serum. Shake gently and allow the 
tubes to stand for 24 hours at room temperature. 



"Medizin. Klinik, vol. 7 (1911), 60. 



SERODIAGNOSTIC METHODS. 41 



The formation of a distinct precipitate indicates 
a positive reaction. A slight cloudiness has no 
significance. 

H. and P. found the test slightly more delicate 
than the Wassermann. Jensen and Feilberg 14 sug- 
gest that the tubes be allowed to stand in an incu- 
bator at 21°, instead of at the somewhat uncertain 
room temperature, and that the two solutions be 
mixed together in equal volumes just before use, 
and then combined in equal volumes with the 
serum. They found the test slightly less delicate 
than the Wassermann reaction. 

The Agglutination Reactions. 

Principle. — When a homogeneous, i.e., uni- 
formly cloudy, suspension of bacteria is mixed 
with the homologous immune serum, a precipitate 
develops after a short time, which consists of 
bacteria clinging to one another, and which sinks 
slowly to the bottom with clearing of the super- 
natant fluid. Motile species lose their motility. 

Practical Applications. — The identification of 
unknown species of bacteria recovered from de- 
jecta, water, etc. (the Gruber-Widal reaction) ; 
e.g., the B. Typhosus, Paratyphosus, Dysenteries, 
Bubonis, V. Cholera Asiatics, D. Meningitidis, 
etc. 



"Berl. klin. Woch., vol. 49 (1912), 1086. 



42 SERODIAGNOSTIC METHODS. 



The Macroscopic Reaction (Method of 
Proscher). 15 — Apparatus. — (a) A homogeneous 
suspension of the bacterium to be identified; for 
this purpose a 12- to 24-hour-old bouillon culture 
is usually sufficient. Thread- and sediment-form- 
ing varieties are better grown on agar slants at 
37° C, and, when 12 to 24 hours old, mixed as 
uniformly as possible, with a platinum loop, in 
a little bouillon. The coarser particles can be 
broken up by rubbing them against the wall of the 
test-tube. 

The suspension should be as thick as a luxuri- 
antly grown bouillon culture. As a measure of 
self -protection the bacteria may be killed by add- 
ing 0.5 per cent, carbolic acid (1-10 volume of 
5 per cent, carbolic acid) before proceeding to the 
test. 

(b) A highly potent immune serum. This is 
most easily obtained by giving rabbits several 
injections of bacteria killed by heating for one 
hour at 60° C. The injections may be intravenous 
or intraperitoneal. The interval between injec- 
tions should be about 7 days. The blood is with- 
drawn 7 to 10 days after the last injection. 

The serum may be kept in the dark, in a cool 
place, 0.5 per cent, carbolic acid being added as a 
preservative. It is better, however, to dry it in 
vacuo, and to seal it in brown glass vials contain- 
ing 0.2 gramme to the vial. 

15 Zentralbl. f. Bakt., etc., vol. 31 (1902). 



SERODIAGNOSTIC METHODS. 43 



(c) Small test-tubes, about 10 cm. long by 
9 mm. inside diameter. 

(d) Volumetric pipettes, 1 c.c. in 1-100 's for 
the preparation of dilutions ; and 1 c.c. and 10 c.c. 
pipettes in 1-10 's for the addition of the sodium 
chloride solution and bacterial emulsion. The 
calibration should extend to the point of the 
pipette. 

(e) Physiological salt solution. 

(/) In place of test-tubes, small glass dishes, 
such as are used for staining microscopic prepara- 
tions, may be used. 

(g) A microscope with dry lenses magnifying 
about 50 diameters. 

Technique. — The following dilutions of the im- 
mune serum are prepared with physiological salt 
solution : 

1-50 (i.e., 0.1 c.c. serum + 4.9 c.c. salt solution). 
1-100 (i.e., 4 c.c. dilution 1-50 + 4 c.c. salt solution). 
1-500 (i.e., 2 c.c. dilution 1-100 + 8 c.c. salt solution). 
1-1000 (i.e., 1 c.c. dilution 1-100 + 9 c.c. salt solution). 
1-2000 (i.e., 0.5 c.c. dilution 1-100 + 9.5 c.c. salt solution). 

Place 0.5 c.c. of each of these in one of the 
test-tubes above described, and add to each the 
same amount of a homogeneous bacterial emul- 
sion. The quantity of the latter may be varied, 
from 0.2 to 0.5 c.c, according to the richness of the 
suspension. The total contents is then brought to 
1 c.c. by the addition of salt solution. This, of 



44 SERODIAGNOSTIC METHODS. 

course, doubles the dilution of the serum in each 
tube. 

Or, one may transfer direct one loop of bacteria 
from the solid culture medium to each serum dilu- 
tion, and mix as uniformly as possible. 

The controls are : 

(a) The bacterium in question + salt solution 
only, the total volume as above, 1 c.c. 

(b) The highest serum dilution employed + a 
24-hour culture of the bacterial species with which 
the unknown bacterium is sought to be identified, 
and which is homologous with the immune serum 
employed. 

Place all the tubes for 1 to 3 hours in the incu- 
bator at 37° C, and examine with the naked eye, 
or under slight magnification. Control (a) should 
remain homogeneous, while (b) and the test 
proper should show distinct clumping if the reac- 
tion is positive. 

According to Proscher, the test-tubes contain 
ing the several mixtures of serum and bacteria 
may be poured into small staining dishes, and 
these may be nested on top of one another, set in 
the incubator, and, after the proper interval, ex- 
amined with the microscope under a magnification 
of about 50 diameters, the condenser being re- 
moved for the purpose. (The fact that cultures 
of Typhosus, etc., recently isolated from water, 
stools, blood or urine are apt to be much less 
agglutinable than laboratory stock cultures of the 



SERODIAGNOSTIC METHODS. 45 



same species, renders this and the following test 
of doubtful value, unless great judgment is used 
in the interpretation of the results. — Translator,) 
The Microscopic Reaction (Hanging Drop 
Method.). — When, to save time, one wishes to test 
colonies taken direct from a plate, the test must 
be made in a hanging drop, on account of the small 
amount of bacterial growth available. Appa- 
ratus. — (a) Hollow ground slides. 

(b) Cover glasses. 

(c) Vaseline, with which to ring the edge of the 
depression in the slide, and thus secure exclusion 
of air from the moist chamber. A brush is con- 
venient to handle the vaseline with. 

(d) Immune serum of high potency, as de- 
scribed on page 42. 

(e) Physiological salt solution. 

(/) Pipettes as described under (d)> p. 43. 

(g) A platinum loop and a platinum needle. 

(h) Microscope with homogeneous immersion 
lens. 

Technique. — One arranges dilutions of the im- 
mune serum as for the macroscopic test. For 
example : 

1-100 (i.e., 0.1 c.c. serum + 9.9 c.c. salt solution). 
1-500 (i.e., 2 c.c. dilution 1-100 + 8 c.c. salt solution). 
1-1000 (i.e., 1 c.c. dilution 1-100 + 9 c.c. salt solution). 

If the amount of bacterial growth in the colony 
to be tested is very slight, one must be satisfied 
with a single dilution, viz., 1-100. 



46 SEKODIAGNOSTIC METHODS. 



With the platinum loop, place the smallest 
possible drop of each of these dilutions in the 
centre of a thoroughly clean cover glass; with 
the platinum needle material is removed from the 
desired colony, and rubbed up to a homogeneous 
emulsion, till a distinct cloudiness is visible to the 
naked eye. (Caution: avoid scattering droplets 
of the emulsion.) The hollow ground slide, pre- 
viously ringed with vaseline, is pressed upon the 
cover glass with the depression down in such a 
manner that the drop lies exactly in the middle 
of the depression. The whole preparation is then 
turned over. The cover glass now lies on the top, 
with the drop hanging in the depression. If the 
drop touches the lower wall of the moist chamber 
the drop is too large, and the preparation must 
be discarded. 

When the serum used is very strong, agglu- 
tination, (i.e., immobilization of motile varieties, 
and clumping) may be observed immediately after 
mixing the serum with the bacteria, or, at the most, 
after twenty minutes ' incubation. Clumps found 
at the periphery of the drop, while the bacteria at 
the centre remain free, have no significance. 

DETERMINATION OF THE AGGLUTINATING POWER OF A 

patient's SERUM (the widal reaction). 
The Macroscopic Method of Proscher. — Ap- 
paratus. — (a) At least 0.1 c.c. of the patient's 
serum. 



SERODIAGNOSTIC METHODS. 47 



(b) A homogeneous suspension of the species 
of bacteria suspected to have caused the disease 
(B. Typhosus, Paratyphosus, Dysenterice, V. 
Cholerce, etc.). When a large number of tests is 
to be made, the emulsion may be prepared in large 
amounts by inoculating a litre, for example, of 
bouillon; after growing for 24 hours, it may be 
killed by the addition of 0.5 per cent, carbolic acid ; 
or mass cultures may be prepared on agar in 
broad, shallow flasks, and after 24 hours washed 
off with carbolized salt solution. The emulsion is 
made as uniform as possible by shaking. Such an 
emulsion may be used for months if kept in a 
cold place. Since the bacteria gradually settle on 
standing, the emulsion should be thoroughly 
shaken before use. The emulsion is made more 
stable by adding about 10 per cent, milk sugar 
in addition to the carbolic acid. Emulsions which 
agglutinate spontaneously can not, of course, be 
used. 

For the diagnosis of typhoid and paratyphoid 
fever, one may use with advantage the typhoid or 
paratyphoid diagnosticum of Ficker as prepared 
by Merck (and others, Trans.), which contains the 
essential parts of the bacterial cell, and remains 
usable for months. The sediment which forms 
on standing should be removed by shaking before 
use. 

For the preparation of small quantities of 
emulsion see above (a), p. 42. 



48 SERODIAGNOSTIC METHODS. 



(c) Small test-tubes as above (c), p. 43. 

(d) Volumetric pipettes as above (d), p. 43. 

(e) Physiological salt solution. 
(/) Small glass watch crystals. 

(g) Microscope, with a dry lens magnifying to 
about 50 diameters. 

Technique. — The patient's serum is first 
diluted 10 times. For this purpose it is drawn 
up in a volumetric pipette, measured, placed in a 
test-tube, and 9 volumes of salt solution added. 
A row of test-tubes is then arranged in a suitable 
rack. In each tube, omitting the first, place 0.5 c.c. 
salt solution. In tubes 1 and 2 place 0.5 c.c. of the 
1-10 patient's serum. In tube 3 place 0.5 c.c. from 
tube 2 ; in tube 4, 0.5 c.c. from tube 3, etc. After 
each addition the tubes should, of course, be thor- 
oughly mixed, and the volumetric pipette employed 
should be thoroughly rinsed out before proceeding 
to the next transfer, unless a fresh pipette is used 
each time. The tubes will finally contain each 
0.5 c.c. of serum diluted 1-10, 1-20, 1-40, etc. 
Usually five or six dilutions are employed. 

The control is a tube containing no serum, but 
only 0.5 c.c. salt solution. 

Now add 0.3 to 0.5 c.c. of the bacterial emulsion 
to each tube, and, if necessary, bring all tubes to 
1 c.c. by adding salt solution. The same amount 
of emulsion should be added to each tube. The 
serum dilution is thus doubled in each tube. The 



SERODIAGNOSTIC METHODS. 49 



following table explains how the several dilutions 
are reached : 





Tube 


Tube 


Tube 


Tube 


Tube 


Tube 


Tube 




No. 1. 


No. 2. 


No.,3. 


No. 4. 


No. 5. 


No. 6. 


No. 7. 


Salt solution . . 


0 


0.5 


0.5 


0.5 


0.5 


0.5 


0.5 


Serum, 1-10. . . 


0.5 


0.5 


0 


0 


0 


0 


0 


From tubeNo.2 


0 


0 


0.5 


0 


0 


0 


0 


From tubeNo.3 


0 


0 


0 


0.5 


0 


0 


0 


From tubeNo.4 


0 


0 


0 


0 


0.5 


0 


0 


From tubeNo.5 


0 


0 


0 


0 


0 


0.5 


0 


From tubeNo.6 


0 


0 


0 


0 


0 


0.5* 


0 



Final dilutions after bacterial emulsion is added are 1-20, 1-40, 1-80, 
1-160, 1-320, 1 640, control. 



* Discarded. 

The rest of the test is carried out as described 
on p. 43. 

"When the Ficker Diagnosticum is employed the 
tubes remain at room temperature, and should 
show after 10 to 14, or, at the most, 20 hours, clear- 
ing of the fluid, with the formation of a precipitate. 

Experience has shown that the reaction should 
be regarded as positive only when agglutination 
occurs in dilutions of 1-40, 1-80, or higher. In 
doubtful cases the test should be repeated every 
2 or 3 days, since the agglutinin content of the 
serum is usually found in increasing amounts in 
typhoid. In the presence of icterus no significance 
attaches to reactions in dilutions less than 1-100, 
since in this condition positive reactions have often 
been observed in dilutions up to 1-75, in the 
absence of typhoid. 

4 



50 SERODIAGNOSTIC METHODS. 



Since it is especially important, for clinical 
purposes, to determine to which one of the typhoid- 
like organisms the disease is due, it is recom- 
mended that the test be carried out not only with 
the typhoid bacillus, but also at the same time with 
the types A and B of the paratyphoid bacillus, or 
with the corresponding diagnostica of Ficker. 

Method of Kafka. 16 — In addition to the patient's 
serum, there are required for this method : 

1. Small staining dishes to hold about 2 or 3 c.c. 

2. A capillary pipette, made from glass tubing 
of about 5 mm. diameter, drawn out and calibrated 
empirically in such a manner that it divides 0.5 c.c. 
into 40 drops. 

3. Ficker 's Typhoid and Paratyphoid Diag- 
nostica. 

4. A volumetric pipette, 5 c.c. divided in 1-10 's. 

Technique. — Place 0.5, 1, and 2.5 c.c. respec- 
tively of each diagnosticum in the staining dishes 
and with the capillary pipette add 1 drop of serum 
to each. The serum is thus diluted 1-40, 1-80, and 
1-200. Mix thoroughly by tipping the dishes back 
and forth, and allow them to stand at room tem- 
perature. The results are observed at the end of 
24 hours. For the diagnosis of an existing typhoid 
infection (but not of an infection of some time 
previous), the reaction must occur in a dilution of 
1-200, while the paratyphoid shows a titre more 



16 Zentralbl. f. Bakt., vol. 40 (1906). 



SERODIAGNOSTIC METHODS. 51 



or less lower than this. If two or more of the diag- 
nostica are positive in the highest dilution, the 
serum must be further diluted, and the test re- 
peated till a higher titre for one is found than 
for the other two. The following table illustrates 
how various dilutions may be obtained : 

4 drops serum 1-10 + 0.5 c.c. diagnosticum = dilution 1-100 
2 drops serum 1-10 + 0.5 c.c. diagnosticum = dilution 1-200 
1 drop serum 1-10 + 0.5 c.c. diagnosticum = dilution 1-400 
1 drop serum 1-10 + 1.0 c.c. diagnosticum — dilution 1-800 
1 drop serum 1-10 + 1.5 c.c. diagnosticum = dilution 1-1200 
1 drop serum 1-10 + 2.0 c.c. diagnosticum = dilution 1-1600 
1 drop serum 1-10 + 2.5 c.c. diagnosticum = dilution 1-2000 
et csetera. 

The diagnosticum which reacts in the highest 
dilution may, with however, some reserve, be re- 
garded as indicating the etiologic agent. If the 
titre for the typhoid is the same as that for the 
paratyphoid, or even if the latter is but slightly 
greater, the typhoid should still be regarded, ac- 
cording to Guttler, 17 as the etiologic factor. (It is 
doubtful if a differential diagnosis between ty- 
phoid and paratyphoid fever can be made by this 
or any similar method. The quantitative relation 
of the direct to the group agglutinins is so irregu- 
lar, and the relative agglutinability of different 
strains of the bacteria concerned so variable, that 
such comparative quantitative tests can hardly 
amount to more than the roughest of approxi- 
mations. — Trans.) 

"Berl. klin. Woch. (1904), Nos. 51 and 52. 



52 SERODIAGNOSTIC METHODS. 



Hanging-Drop Method. -Apparatus. — (a) The 
serum to be tested. 

(b) Cover glasses. 

(c) V aseline and brush. 

(d) Hollow ground slides. 

(e) Physiological salt solution. 

(/) Volumetric pipettes, 0.1 c.c. in 1-100 's, and 
10 c.c. and 1 c.c. in 1-10 's. 

(g) Microscope with oil immersion lens. 

(h) A 10- to 24-hour-old agar slant culture of 
the organism against which the serum is to be 
tested (typhoid, paratyphoid, etc.). 

(i) Platinum needle and platinum loop. 
Technique. — The technique is that given on 

p. 45, with the following exception : 

Prepare the following dilutions of the patient's 
serum : 

1-25 = 0.1 c.c. serum + 2.4 c.c. salt solution. 
1-50 == 1 c.c. dilution 1-25 + 1 c.c. salt solution. 
1-100 = 0.5 c.c. dilution 1-50 + 0.5 c.c. salt solution. 
1-150 = 0.5 c.c. dilution 1-50 -f- 1 c.c. salt solution. 

Mix a trace of the culture, by means of the 
platinum needle, with a drop of each of these dilu- 
tions. The controls are: (1) A drop of bouillon 
or salt solution + bacteria as above, and (2) a 
drop of known positive agglutinating serum + 
bacteria. When the reaction is positive, agglu- 
tination occurs in the dilutions of the patient's 
serum up to 1-100 or higher, and in the second 
control, while the first control remains unchanged. 



SERODIAGNOSTIC METHODS. 



53 



Diagnosis of Mixed Infection by Means of the 
Agglutination Test (The Saturation Test of 
Castellani). 18 — If the agglutinating power of a 
patient's serum is tested against both the homol- 
ogous organism and against closely related 
organisms {e.g., typhoid and paratyphoid bacilli), 
an increased agglutinating power against the lat- 
ter as well as the former is frequently found, 
although it has no share in the production of the 
disease. This is the so-called " group agglu- 
tination. ' ' 

On the other hand, in case of mixed infection, 
the agglutinating power is likewise increased 
against the several species of organisms con- 
cerned. In order to determine, in a given case, in 
which a serum agglutinates several species 
equally, whether one is dealing with a true mixed 
infection, or whether the result is due to group 
agglutination merely, the Castellani test may be 
employed. 

Principle. — (A) The serum of an individual 
immunized against a single species only loses its 
agglutinating power for all the species it is capable 
of influencing, when mixed with an excess of the 
homologous organism, which absorbs the entire 
mass of agglutinins; but when mixed with other 
than the homologous organism, i.e., with organ- 
isms influenced only through group agglutination, 



,8 Zeitschr. f. Hyg., vol. 40 (1902). 



54 SERODIAGNOSTIC METHODS. 



it loses its agglutinating power for these organ- 
isms only, but not for the homologous organism. 

(B) The serum of an individual immunized 
against two organisms, A and B, loses its agglu- 
tinating power for A only, when mixed with an 
excess of A; and for B only when mixed with an 
excess of B. 

The apparatus required is essentially that 
needed for the Widal test, as previously described. 

Technique. — From 0.5 c.c. to 1.0 c.c. of the 
serum to be tested, and which, we will assume, 
agglutinates equally the typhoid and colon bacilli, 
is placed in each of two test-tubes, and mixed with 
relatively large amounts of the t} r phoid and colon 
bacillus respectively (4 to 8 loops of a 24-hour-old 
agar slant culture). The bacteria are thoroughly 
mixed with the serum, and the tubes then placed 
in the incubator for 12 hours. At the end of this 
time the serum, after being separated from the 
agglutinated masses of bacteria by centrifugation, 
is tested to determine whether it still contains 
agglutinating power for the same organism as that 
first added. If so, more bacteria are added, and 
the incubation is repeated. When the agglutinat- 
ing power for the respective organism employed 
has disappeared (in which case the emulsion re- 
mains cloudy), the bulk of the bacteria remaining 
is removed by centrifugation at a high speed, or 
by allowing the tubes to settle for 12 to 24 hours 
in the ice-box. The serum is then tested for its 



SERODIAGNOSTIC METHODS. 55 



agglutinating power against the species which was 
not used in the first test. Thus if the typhoid bacil- 
lus was added in the first instance to tube A, the 
serum in this tube is now tested against the colon 
bacillus; and tube B, to which the colon bacillus 
was first added, is now tested against the typhoid 
bacillus. 

Should the test show that absorption of agglu- 
tinins by each species leaves the agglutinating 
power for the other practically unchanged, the 
case is in all probability one of mixed infection by 
the typhoid and colon bacilli. If on the other hand 
absorption of agglutinin by the typhoid bacillus 
causes a material reduction in the agglutinating 
power for the colon bacillus, it is probably an in- 
fection by the typhoid bacillus alone, and the effect 
of the serum on the colon bacillus is to be regarded 
as due merely to group agglutination. 

Practical tests of the Castellani method are not 
yet very numerous. 

Bactericidal Eeactions. 
In the Living Animal (the Pfeiffer Phenome- 
non). — Principle. — When bacteria, mixed with the 
homologous immune serum, are injected into the 
peritoneal cavity of a guinea pig, solution of the 
bacteria occurs in a short time, with the typical 
granule formation; but when injected along with 
a normal or heterologous immune serum, they re- 
main intact, or even actively motile. 



56 SERODIAGNOSTIC METHODS. 



PKACTICAL APPLICATIONS. 

Identification of Bacteria Recovered from the 
Dejecta, Water Supplies, etc — (The method is chiefly 
employed in the identification of the cholera and 
similar vibrios, typhoid, and paratyphoid bacilli, 
etc.). 

Apparatus. — (a) Four guinea pigs, weighing 
from 200 to 300 grammes each. 

(b) Immune serum of high potency. This is 
most easily obtained by giving rabbits one or more 
intraperitoneal injections of a known culture of 
the organism with which the unknown organism is 
to be identified. The organism should be grown 
on agar, an emulsion prepared and killed at 60° C. 
The animal should be killed 14 days after the last 
injection. 

The mixed serum of several animals so treated 
should be dried in vacuo, and placed in small 
amber colored glass vials, containing 0.2 gramme 
each, for preservation. Such vials may be ob- 
tained from the Bacteriological Institute of the 
Royal Prussian Institute for Infectious Diseases. 

The serum must be at least of such a strength 
that 0.0002 gramme suffices to convert completely 
within one hour 1 loop (=2 mg.), of an 18-hour- 
old culture of a virulent cholera vibrio or typhoid 
bacillus respectively, into granules when injected, 
mixed with 1 c.c. of bouillon, into the abdominal 



SERODIAGNOSTIC METHODS. 57 



cavity of a guinea pig. In other words, its ' ' titre ' ' 
must be at least 0.0002 gramme. 

(c) Eabbit serum, fresh, or dried and pre- 
served in the same way. 

(d) An 18-hour-old agar slant culture of the 
organism to be tested. 

(e) A syringe with a blunt needle. 

(/) Shears and forceps for making the incision 
for the injection. 

(g) Several capillary tubes, prepared by draw- 
ing out glass tubing 0.5 cm. in diameter. These 
are used for removing the fluid from the abdom- 
inal cavity. 

(h) Four funnel-shaped glasses, of the size and 
shape of a liqueur glass, in which to mix the fluids 
for injection. 

(i) Hollow ground slides, cover glasses, and 
vaseline for the preparation of hanging drops. 

(k) A platinum loop calibrated to take up 
2 mg. of the bacterial growth. 

(I) Volumetric pipettes, 0.1 c.c. in 1-100's and 
1 c.c. and 10 c.c. in 1-10 's. 

(m) Sterile bouillon. 

(n) Physiological salt solution. 

Technique. — In glass 1 place five times the titre 
unit of the serum (i.e., about 0.001 gramme of a 
serum having a titre of 0.0002). 

In glass 2 place ten times the titre unit of the 
serum (0.002 gramme in our example). 

In glass 3 place, as a control, fifty times the 



58 SERODIAGNOSTIC METHODS. 



titre unit of normal serum of the species furnish- 
ing the immune serum. 

In order that the various amounts of serum 
may have like relations, they must be contained 
in equal volumes of fluid. The amounts of serum 
mentioned above are therefore prepared in suit- 
able dilutions. In our example, 0.2 gramme dried 
immune serum is dissolved in 10 c.c. salt solution. 
Of this 0.2 c.c, representing 0.002 gramme, or 
2 mg., is placed in the second glass. A portion 
of the remaining fluid is then mixed with an equal 
volume of salt solution, so that the 0.2 c.c. to be 
placed in the first glass, represents 1 mg. of 
serum. The required amount of normal serum is 
obtained by dissolving 0.2 gramme of the dried 
serum in 4 c.c. of salt solution. 

Finally, in glass 4, place 4 c.c. of bouillon, 
0.8 c.c. salt solution, and 1 loop from the agar 
culture. The 1.2 c.c. injected will thus contain 
exactly .25 loop of bacteria. 

The contents of the various glasses having been 
thoroughly mixed, the injection is made, by incis- 
ing the outer skin with the scissors, and puncturing 
the peritoneum with the dull needle. The first 
three guinea pigs receive the contents of the first 
three glasses respectively, plus 1.2 c.c. from the 
fourth glass. The fourth animal receives only 
1.2 c.c. from the fourth glass. 

At the end of 20 minutes, and again at the end 
of an hour, a drop of fluid is removed from the 



SERODIAGNOSTIC METHODS. 59 



abdominal cavity of each animal, through the same 
opening, by means of one of the capillary pipettes 
(usually pressure on the abdomen suffices to cause 
the fluid to enter the tube), and examined in a 
hanging drop preparation. 

If guinea pigs Nos. 1 and 2 show granule for- 
mation, at the latest after an hour, while in the 
third and fourth animals, large masses of bacteria 
remain motile, and if well preserved vibrios are 
still present, the reaction may be regarded as posi- 
tive, and the diagnosis as established. 

Demonstration of Bactericidal Antibodies in 
the Serum of Sick or Convalescent Patients. (Diag- 
nosis of a recent cholera infection, etc.). — Appa- 
ratus. — The same apparatus is required as on 
p. 56, except that in place of (d) one employs 
an 18-hour-old agar slant culture of the true 
cholera vibrio, or typhoid bacillus, as the case may 
be ; and in place of ( b ) , the serum of the patient. 

Technique. — Dilutions of the patient's serum 
are prepared with 20, 100, and 500 parts of sterile 
bouillon, and 1 c.c. of each of these is placed in one 
of the first three liqueur glasses. To each glass 
is then added 1 loop of the culture. 

Glass four contains 4 c.c. of bouillon without 
any serum, + 1 loop of culture, so that the amount 
injected (1 c.c), contains .25 of a loop. The re- 
maining technique is as above, p. 58. 

If granule formation occurs in the first three 
animals, but is wanting in the fourth, the reaction 



60 SERODIAGNOSTIC METHODS. 



may be regarded as positive, and the diagnosis of 
cholera, etc., as established. 

If granule formation occurs in the fourth ani- 
mal also, the culture employed is to be regarded 
as unsuitable. 

Bactericidal Reactions in Vitro (Determination 
of Bactericidal Substances in the Blood-Serum of 
Patients). — This method was devised by Stern 
and Korte, 19 for the diagnosis of typhoid fever. 
It is complicated, and is only rarely to be recom- 
mended, in cases in which the agglutination test 
has yielded uncertain results. 

Principle. — The patient's serum is inactivated 
by heating for half an hour at 56° C, and is then 
mixed with a fresh complement containing normal 
serum, which is, per se, inactive. "Plating out" 
tests are then made to determine in how great 
a dilution of the serum bactericidal activity can 
still be demonstrated, as shown by a reduction in 
the number of colonies as compared with control 
tests. 

Apparatus. — (a) The patient's serum, heated 
for half an hour at 56° C, and then diluted 50 to 
100 times with sterile, 0.85 per cent, sodium chlo- 
ride solution. 

(b) Serum of a normal rabbit, obtained the 
same day it is to be used, and diluted 10 to 15 
times, to serve as complement. 



19 Berl. klin. Woch. (1904), 



SERODIAGNOSTIC METHODS. 61 



(c) A 24-hour-old bouillon culture of typhoid 
bacilli, diluted 500 times with physiological salt 
solution. 

(d) Short, sterile test-tubes (about 10 cm. 
long) , of the usual diameter, provided with cotton 
plugs, and arranged in a rack. 

(e) Sterile physiological salt solution. 
(/) Sterile Petri dishes. 

(g) Nutrient agar, in tubes. 

(h) Sterile pipettes, 10 c.c. and 1 c.c. in 1/10 's. 

(i) A water bath for melting the agar. 
(j) A thermometer. 

(k) An incubator at 37° C. This may be dis- 
pensed with if necessary. 

Technique. — Place 1 c.c. of sterile salt solution 
in each of 12 test-tubes, arranged in a rack. Then 
place in the first of these tubes 1 c.c. of the inacti- 
vated and diluted patient's serum. Mix thor- 
oughly by repeated sucking up and forcing out 
again of the fluid; and then (with the same 
pipette) draw up 1 c.c. and transfer to the second 
tube of the series. Mix as before, and transfer 
1 c.c. from this tube to the third. Continue thus 
to the last tube, from which, finally, 1 c.c. is 
discarded. 

Each tube now contains 1 c.c. fluid, represent- 
ing dilutions of 1-2, 1-4, 1-8, 1-16, etc., of the 
original dilution of the patient's serum. 

Now add to each tube 0.5 c.c. of the bacterial 



62 SERODIAGNOSTIC METHODS. 



emulsion; and finally, 0.5 c.c. of the diluted com- 
plement serum. 

The controls are l-(a) and (b) each 1.5 c.c. 
of salt solution + 0.5 c.c. of the bacillary 
emulsion ; 2-1 c.c. salt solution + 0.5 c.c. diluted 
complement serum + 0.5 c.c. bacillary emulsion. 

Control tube (1 a) is immediately mixed with 
agar, previously liquefied and cooled to 41° C, 
rapidly mixed, and poured into a sterile Petri 
dish. This plate shows the original number of 
colonies. 

All the remaining tubes are placed for from 
3 to 4 hours in the incubator, and are thereafter 
plated in the same manner as (1 a). Gelatine 
may be used in place of the agar if desired. 

Control (lb) will show how much the bacteria 
increased during the period of incubation, in the 
absence of bactericidal substances. Control 2 
shows the lack of activity of the complement 
serum alone, in the absence of the specific bac- 
teriolysin. If, contrary to expectation, this con- 
trol shows marked bactericidal activity, the entire 
test is without significance. The plates, which 
are to be placed in the incubator bottom up, may 
be examined after as short a time as twelve hours. 
(Gelatine plates, which must be kept at 22°, are 
ready for examination only after 48 hours.) 

It is unnecessary actually to count the colon- 
ies which develop, since significance can only be 
attached to marked and immediately recognizable 



SERODIAGNOSTIC METHODS. 63 



differences. As an example, an experiment of 
Stern and Korte, with a highly potent typhoid 
serum is here reprinted. 



1 c.c. typhoid 
serum. 



Typhoid 
culture. 



Rabbit serum 
1-12. 



Plates poured after three 
hours at 37°. 



1-2000. . . 
1-4000. . . 
1-8000. . . 
1-16000. . 

1-32000. . 
1-64000. . 
1-128000. 
1-256000. 

1-512000. 
1-1024000 
1-2048000 
1-4096000 



0.5 
0.5 
0.5 
0.5 

0.5 
0.5 
0.5 
0.5 

0.5 
0.5 
0.5 
0.5 



0.5 
0.5 
0.5 
0.5 



0.5 
0.5 
0.5 
0.5 



0-100 colonies 



100-1000 colonies 



More than 1000 colonies 
Less than in control 2 
Less than in control 2 
Like control 2 



Controls: 

(la) 1.5 c.c. NaCl + 0.5 typhoid culture poured 
at once 35,000 colonies 

(1 6) 1.5 c.c. NaCl + 0.5 typhoid culture poured 
after 3 hours 00 colonies 

(2) 1.0 c.c. NaCl + 0.5 typhoid culture + 0.5 rab- 
bit serum 1-12, poured after 3 hours 00 colonies 

According to Hahn, 20 the sera of non-typhoid 
individuals show bactericidal effects in only about 
1-3 of the cases. Moreover this is only very ex- 
ceptionally demonstrable in dilutions above 
1-500. On the other hand, the sera of patients 
still ill with the fever, or but recently recovered, 



^Deutsch. Arch. f. klin. Med. (1905). 



64 SERODIAGNOSTIC METHODS. 



are bactericidal in dilutions greater than 1-1000, 
and usually as high as 1 in 50,000. (In the experi- 
ment recorded on page 48, this effect is manifest 
in a dilution of 1 in 2,048,000, and is marked in a 
dilution of 1 in 512,000.) 

The higher the bactericidal titre runs above 
that usually found in normal sera, the more prob- 
able it is that the case is one of typhoid infection. 

Cytolytic Eeactions 

Cancer Diagnosis of Freund and Kaminer. 21 — 

Principle. — The blood-serum of normal individ- 
uals has the power to dissolve cancer cells. The 
blood-serum of patients suffering from cancer 
lacks this power, and has the power to inhibit the 
destruction of such cells by normal serum. 

When the blood-serum of cancer patients is 
mixed with a cancer extract, a precipitate forms. 

Application. — The diagnosis of malignant 
disease. 

Apparatus. — (1) An emulsion of cancer cells. 
Grind in a mortar the undegenerated portions of 
the tumor, freed so far as possible of fat and 
extraneous cells, with about five volumes of 1 per 
cent, sodium biphosphate. Squeeze the suspen- 
sion through several thicknesses of gauze. Allow 
the cells to settle, and remove the supernatant 

21 Biochem. Zeitschr., 1910; Wien. klin. Woch., vol. 23 (1910), 
378 and 1221; 24 (1911), 1759. 



SERODIAGNOSTIC METHODS. 65 



fluid with a pipette. Wash the residue with 0.6 
per cent, sodium chloride solution. Allow the 
cells to settle again, pipette off the supernatant 
fluid and cover the residue with one per cent, 
sodium fluoride. The last named fluid should be 
first neutralized against alizarin till only a trace 
of violet color remains. This emulsion will keep 
for several weeks in the ice-box. 

(2) An extract of cancer cells. Fresh tumor 
tissue obtained at autopsy, or tumor tissue pre- 
served in alcohol may be used. Portions as free 
as possible of degenerated areas and fat are cut 
into small pieces and worked through a coarse 
meshed cloth by the gradual addition of 10 vol- 
umes of 0.6 per cent, acid sodium phosphate solu- 
tion. Allow the cells to separate by settling or by 
careful centrifugation. Wash several times with 
the same fluid, and preserve at 0°. Sodium fluoride, 
up to 1 per cent., may be added as a preservative, 
provided care is taken to see that it does not 
render the fluid alkaline. Thymol may also be 
used. 

For use add 5 c.c. of 5 per cent, acetic acid to 
100 c.c. of the fluid. Heat the mixture in the 
water-bath for 15 minutes at 80°, filter, cool, and 
neutralize to litmus with sodium carbonate. Heat 
again as above, cool and filter. Heating at 100° 
or over the free flame must be avoided. The activ- 
ity of the extract is determined by testing with 
10 drops of known normal and cancerous sera 
5 



66 SERODIAGNOSTIC METHODS. 



whether the extract, undiluted, and diluted 10, 
50, and 100 times, gives a precipitate with cancer 
serum, plainly visible in test-tubes held against 
the window, while the normal serum gives no such 
precipitate. The extract keeps for only two or 
three days. The extract if used in too concen- 
trated form causes a precipitate with normal 
serum, but if diluted to opalescence gives a dis- 
tinct cloud which clears upon the addition of nor- 
mal serum, or of an ether extract of horse serum. 

(3) The patient's serum. This is obtained in 
any of the customary ways. The serum must be 
separated from the cells within a few hours after 
the blood is drawn and cannot be used when it is 
more than 48 hours old. 

(4) A blood-counting chamber. 

(5) Small test-tubes or small test dishes with 
parallel sides. 

(6) Capillary pipettes, and volumetric pi- 
pettes, 1 c.c. in 1-100 's. 

(7) An incubator at 37°. 

(8) An 0.5 per cent, solution of sodium 
fluoride. 

Technique. — To 10 drops of the patient's 
serum add 1 drop of the 0.5 per cent, solution of 
sodium fluoride. Then add 1 drop of the cancer 
cell emulsion so diluted that when 1 drop of the 
mixture is placed in the blood-counting chamber, 
about 10 to 20 tumor cells will be found in a large 
field (25 of the smallest squares) of the apparatus. 



SERODIAGNOSTIC METHODS. 67 



Close the counting chamber carefully and place 
in the incubator for 24 hours. Count the number 
of cells again. 

A material reduction in the number of cells 
will be found when the serum is derived from a 
healthy individual, whereas, if the serum is de- 
rived from a cancer patient such a reduction does 
not take place. 

A second test is made by diluting normal 
serum with an equal amount of 0.6 per cent, so- 
dium chloride solution, and also with an equal 
volume of the patient's serum. To each of these 
fluoride and the cell emulsion is added as above. 
If the patient's serum prevents the solution of the 
cells by the normal serum, this again constitutes 
a positive reaction. 

The test for a precipitate is made as follows : 
Place 10 drops of the patient's serum in a small 
test-tube or test dish with parallel sides and add 
2 c.c. of the extract properly diluted as described 
above. Controls are made by adding to a sepa- 
rate quantity of serum a fluid identical with the 
tumor extract with the exception of the tumor 
tissue. In other words, if the extract was pre- 
pared from fresh tumor tissue this fluid is pre- 
pared by adding to 100 c.c. of 1 per cent, acid 
sodium phosphate, 5 c.c. of 5 per cent, acetic acid, 
and neutralizing with sodium carbonate. When 
the extract is prepared from tissues preserved in 



68 SERODIAGNOSTIC METHODS. 



alcohol, use 0.25 per cent, acetic acid neutralized 
with sodium carbonate. 

The precipitate forms at once and must be 
viewed by transmitted, not by reflected light. A 
distinct clouding under these conditions consti- 
tutes a positive reaction. The authors of the test 
recommend that all three tests should be made 
when enough serum is available. This calls for 
about 2.5 c.c. of serum, representing about 6 c.c. 
of whole blood. 

Diagnosis of Pregnancy (Improved Method of 
Abderhalden). 22 — Principle. — The blood serum of 
pregnant individuals contains a specific ferment 
which digests placental cells. 

Apparatus. — (1) Placental tissue. Cut a fresh 
human placenta into small pieces, place in a wide 
dish, and wash in running (tap) water. The ma- 
ternal portions may be separated and discarded, 
but this is not necessary. While the washing, 
which is for the purpose of removing blood, is 
going on, a large enamel basin or evaporating dish 
containing 1 to 2 litres of water, to which 1 drop 
per litre of glacial acetic acid is added, is heated 
to boiling. Throw the washed placenta into the 
boiling water, and continue the boiling for five 
minutes. Pour off the boiling water through a 



22 Abderhalden, Schutzfermente des tierischen Organismus. 
Berlin, Julius Springer, 1912. Munch, med. Wochen., vol. 59, 
1912, pp. 1305, 1939, 2172. 



SERODIAGNOSTIC METHODS. 69 



gauze strainer or plaited filter, taking care that 
the coagulated bits of placenta remain as far as 
possible in the dish. Pour on more water and boil 
again for five minutes. Now test a portion of the 
water for the biuret reaction as follows : To about 
10 c.c. of the water, add 5 c.c. of a 33 per cent, 
caustic soda solution, mix and layer enough of a 
very dilute (0.25 per cent.) solution of cupric sul- 
phate on top to make a layer 0.25-0.5 cm. deep. A 
red color at the zone of contact constitutes a posi- 
tive reaction. If the process has been carried out 
rapidly, the test will always be negative. 

In place of the biuret reaction for peptone the 
more delicate triketohydrinden hydrate, Hoechst, 
may be used. This gives a blue color with com- 
pounds which have a COOH group and an 
amino acid in the a position. Albumen, 
peptone, polypeptids and animo acids react. 
Place 10 c.c. of the fluid in a large test tube, add 
exactly 0.2 c.c. of a 1 per cent, aqueous solution 
of triketohydrinden hydrate, bring quickly to boil- 
ing and continue the boiling for one minute with- 
out interruption. When the reaction is positive, 
a beautiful violet blue color develops in a short 
time. When negative, the solution remains color- 
less, or takes on a yellowish tone. The use of the 
triketohydrinden hydrate is recommended, but it 
must not be used for the test proper unless the 
placental tissue, as prepared above, has been 
shoiun to react negatively with it. 



70 SERODIAGNOSTIC METHODS. 



If the water in which the placenta is cooked 
gives one or other of these reactions for peptone, 
the boiling must be continued as before till the 
reaction is negative. When this occurs pour the 
last cooking water, together with the placental 
tissue, into a wide-mouthed flask. Cover with a 
layer of toluol, and cork tightly. The entire pro- 
cess requires not more than 1/2 to 1 hour, and 
furnishes material for several hundred tests. 

(2) Extraction shells, S. & S. 579. These 
should be kept under water and a layer of toluol, 
and should be tested for permeability to Witte's 
peptone before use. After being used, they may be 
carefully washed in running water and used again. 
They should never be used dry. 

(3) The patient's serum. This must not be 
shaken. It is recommended to allow about 10 c.c. 
of blood to flow directly into a centrifuge tube. 
As soon as the clot forms centrifugate, pour off 
the serum, and use at once for the test. If the 
serum contains any hemoglobin it cannot be used. 

(4) Small beakers, Erlenmeyer flasks or 
especially designed cylinders of such a size that 
the shells above mentioned fit into them leaving 
not more than 0.25 cm. between the shell and the 
wall. 

(5) Eeagents for the biuret reaction, or 
triketohydrinden hydrate, or both. 

(6) Incubator at 37° C. 

(7) Distilled water. 



SERODIAGNOSTIC METHODS. 71 



Technique. — In one of the extraction shells, 
previously tested as to its permeability, washed, 
and kept as above described, place about 1 gramme 
of the placental tissue, crumbling it between the 
fingers into pieces the size of a wheat grain while 
doing so. Pour about 2 or 3 c.c. of the patient's 
serum over the tissue in the bottom of the shell. 
Add a few drops of the toluol to prevent putre- 
faction. Now hold the shell at the top, and rinse 
the outside thoroughly under the tap. Place the 
shell in one of the beakers, described above, and 
add 15 c.c. of water, outside the shell. A thin 
layer of toluol is placed also on the outside. Con- 
trols should be prepared in the same way, consist- 
ing of the patient's serum alone, and of the pla- 
cental tissue alone respectively. Place in the 
incubator for 12-16 hours. Now remove 10 c.c. of 
the fluid outside of the shell, by means of a pipette 
thrust below the level of the toluol, and test this 
for peptone with one or both of the reagents 
described above. When triketohydrinden hydrate 
is to be used, it is especially important that as 
little toluol as possible be transferred to the test 
tube, as overheating is then apt to occur. A glass 
rod may be placed in the test tube to prevent 
bumping. 

A positive result is indicated when the dialy- 
sate gives a positive reaction for peptone. 



72 SERODIAGNOSTIC METHODS. 



The method has been verified by E. Franz 23 
and by Frank and Hermann. 24 

The polariscope method of Abderhalden, which 
is given in detail in the monograph first cited 
above, is here omitted because it is so complicated 
as to fall outside the domain of the usual clinical 
laboratory. 

Moro's Alexin Test 25 (Estimation of the Com- 
plement Content of a Patient's Serum.) — Principle. — 
The patient's serum, in increasing quantities, is 
mixed with a fixed quantity of washed erythro- 
cytes, and an amount of inactivated (i.e., comple- 
ment free), immune serum (amboceptor serum), 
sufficient to cause solution of the red cells. After 
incubation for 2 hours, the degree of haemolysis 
is noted. The amount of the patient's serum 
required to bring about haemolysis affords an 
approximate measure of the complement (alexin) 
content of the serum. 

Apparatus. — (1) A 10 per cent, suspension in 
physiological salt solution of washed sheep's 
blood-cells. 

(2) Antisheep immune serum, of high potency, 
and inactivated, to serve as amboceptor. 



23 Franz, Munch, med. Woch., vol. 59 (1912), p. 1702. 

24 Frank and Heimann, Berl. klin. Woch., vol. 49 (1912), 
p. 1706. 

25 Munch, med. Woch. (1907), Nos. 21 and 31, and personal 
communication. 



SERODIAGNOSTIC METHODS. 73 



(3) The serum to be tested, not inactivated. 
About 5 drops drawn from the ear will suffice. 

(4) Volumetric pipettes, in 5-100 's or 1-10's. 

(5) Test-tubes, about 5 mm. inside diameter 
by 5 cm. long. 

(6) A Sahli haemometer. 

Technique. — The hemolytic system is set up 
as follows : 0.1 c.c. sheep blood suspension, + 
0.1 c.c. immune serum diluted 1-500 (titre 1-1000 
to 1-1500), + 0.025 c.c. (0.1 c.c. of a 1 in 4 dilu- 
tion), fresh patient's serum. 

Incubation is followed either by complete 
haemolysis or by incomplete solution, leaving a 
residue of red cells. 

To measure this residue, the fluid is centrif- 
ugated, the supernatant fluid is removed, and the 
sediment, consisting of red-cells, is dissolved in 
0.1 c.c. n/10 HC1. The yellow to brown tint of 
the resulting solution depends upon the trans- 
formation of the haemoglobin into haematin hydro- 
chloride. This fluid is then placed in the gradu- 
ated tube of the Sahli haemometer, and its color is 
compared with that of the standard solution. 

Three findings are possible. 

(1) The color may be paler than that of the 
standard solution. This may be indicated thus, 
n < 0.1, n representing the amount of fluid in 
which the red cell sediment must be dissolved, to 
make it match the shade of the standard solution. 

(2) It may be equal ; n = 0.1. 



74 SERODIAGNOSTIC METHODS. 



(3) It may be darker in color than the stand- 
ard solution. In this case the hsematin solution 
is diluted until the colors match. Should this hap- 
pen, for example, on the addition of 0.25 c.c. of 
water, it is indicated by writing n = 0.1 + 0.25, 
or, for short, 0.35. 

Suppose that in the wholly undissolved con- 
trol, the red cells give a value, thus estimated, 
of 0.8. Then: 

(a) In the complete lack of complement in the 
patient's serum, the resulting value also will 
be 0.8. 

(b) Values between 0.75 and 0.4 indicate a 
more or less markedly reduced complement 
content. 

(c) The normal complement content ranges 
from 0.4 to 0.1. 

(d) Values between 0.1 and 0.0 indicate a 
complement content above normal. 

The following modification is to be preferred, 
as it eliminates the occasionally disturbing in- 
fluence of the native amboceptors for sheep's cells 
sometimes found in human serum. 

0.1 c.c. of a 10 per cent, suspension of washed 
human red cells, + 0.1 c.c. of an appropriately 
diluted antihuman immune serum from a rabbit 
(so diluted that 0.1 c.c. will contain the amount 
needed to dissolve the 0.1 c.c. red cells), + 
0.025 c.c. fresh patient's serum. 

The rest of the test is carried out as above. 



SERODIAGNOSTIC METHODS. 75 



Antih^molytic Reactions. 

complement deviation, of fixation, tests. 

Principle, — Whenever proteid antigens are 
mixed with their homologous specific antibodies, 
in the presence of active, (i.e., complement con- 
taining), normal serum, fixation of the comple- 
ment takes place. This may be recognized by the 
fact that the mixture, in spite of the fact that it 
contains complement, is no longer able to serve 
as complement in bringing about lysis in a hsemo- 
lytic system, consisting of red blood-cells "sensi- 
tized " or charged with a hemolytic amboceptor. 
This complement fixation is specific, and permits 
the recognition of certain antigens, in part of 
bacterial origin, or of their corresponding anti- 
bodies, in the material to be examined; and in 
many respects constitutes a method comparable 
to the precipitin reactions. 

PEACTICAL APPLICATIONS. 

Demonstration of Proteid Antigens (e.g., de- 
termination of human proteid in blood-stains; or 
of bacterial substances in the serum of patients). 

Method of Rickmann. 26 — Apparatus. — (a) A 
serum of high potency, immune to the particular 
antigen to be determined in the material to be 
tested. Its titre must be known. (For the prepa- 
ration see p. 22, Precipitin Reactions.) 



Arbeit, a. d. K. Inst. f. exp. Ther. in Frankfurt, 1907. 



76 



SEEODIAGNOSTIC METHODS. 



(b) Complement containing serum of a nor- 
mal guinea pig, obtained fresh the day it is to be 
used. The simplest procedure is to stun the 
animal by a blow at the base of the skull. Then 
cut its throat and collect the blood in a Petri dish 
or other suitable receptacle. The serum may be 
allowed to separate spontaneously over night in 
the ice-box, or, better, separated at once by centrif- 
ugation. Enough serum to furnish complement 
for several Wassermann tests may be obtained 
from a guinea pig without killing the animal, by 
the following method, modified by the Translator 
from that of Zahn : 27 trim the hair from about 
an ear with scissors. Eub the ear with chloro- 
form or xylol, till the ear becomes hyperamiic. 
Incise one of the larger veins of the ear with a 
sharp scalpel, and fit a small distillation flask 
provided with a side arm as closely as possible 
over the ear. The side arm of the distillation 
flask is connected by means of rubber tubing with 
some form of suction apparatus, such as a 
Chapman pump. If the flask is held in such a 
manner that the side arm points upward, the 
blood will run to the bottom of the flask, as it 
flows from the incision. About 10 c.c. of blood 
can be readily collected in this way. This should 
be poured into a centrifuge tube and centrifugated 
at once to cause separation of the serum. 



27 Munch, med. Wochenschr., vol. 59 (1012), 861. 



SERODIAGNOSTIC METHODS. 77 



The animal may be used repeatedly at inter- 
vals of a week or two. 

(c) A 5 per cent, suspension of sheep or beef 
red blood-cells, washed several times with salt 
solution, to remove serum. To prepare, dilute 
the defibrinated blood with salt solution, cen- 
trifugate, remove the supernatant clear fluid, and 
replace with a like amount of fresh salt solution, 
and again centrifugate. This is repeated at least 
three times. The washed cells are finally mixed 
with twenty volumes of salt solution. 

(d) A perfectly clear solution of the material 
to be examined. (Small blood-stains may be dis- 
solved in the same manner as for the precipitin 
reaction.) 

(e) A known solution of the antigen whose 
presence is to be determined in the material under 
investigation (i.e., horse serum, for example; or, 
if we are dealing with the serum of a typhoid 
patient, then a typhoid bacillus extract). 

Liidke 28 gives the following directions for 
preparing such an extract: A suspension of a 
24-hour-old agar culture is heated for half an 
hour at 60° to kill the organisms, and then 
centrifugated. The sediment, consisting of bac- 
teria, is dried, and placed in a sterile agate mor- 
tar. It is then ground and at the same time 



M Zur Kenntnis der Komplemente, Wiirzburg (1908). 



78 SERODIAGNOSTIC METHODS. 



sterile salt solution is added, drop by drop. More 
salt solution is added after the grinding is com- 
pleted, to form a slightly opalescent fluid. 

Leuchs 29 gives the following directions : Sev- 
eral Kolle's dishes, the surface of each of which 
about corresponds to 10 agar tubes, are inocu- 
lated with the organism desired, and placed in the 
incubator for 24 hours at 37°. The bacteria are 
washed off with sterile distilled water. The sus- 
pension so obtained is placed at 60° for 24 hours 
to kill the bacteria, and is then placed in a shaker 
for 24 hours to bring about autolysis. The sus- 
pension is then thoroughly centrifugated, and the 
clear, supernatant fluid, to which 0.5 per cent, 
phenol is added, is kept in the ice-box, where it 
remains unchanged for at least eight weeks. 

(/) Eabbit's serum of high potency, immune 
to beef or sheep's blood, inactivated by heating 
for a half hour at 56° (amboceptor serum). This, 
like all the fluids used in this test, must be abso- 
lutely clear. 

(g) Test-tubes, 0.9 to 1 cm. diameter by 10 cm. 
long. 

(h) Volumetric pipettes, 0.1 c.c. in 1-100 's 
and 1 c.c. and 10 c.c. in 1-10's. 

(i) Physiological salt solution. 
(j) An incubator at 37°. 



28 Berlin, klin. Woch. ( 1907 ) . 



SERODIAGNOSTIC METHODS. 79 



(k) A water-bath, for inaetivation of the sera. 

Technique. — Previous to the test itself, the 
amboceptor serum, complement serum, and pro- 
teid immune serum must be titrated. 

Titration of the Amboceptor Serum In each 

of a series of test-tubes are placed 1 c.c. of the 
beef blood suspension, 0.1 c.c. of complement 
serum, and increasing quantities of the inacti- 
vated amboceptor serum, somewhat as follows: 



1-100 dilution of the ambocep- 
tor serum, in salt solution. 0.25 c.c. = 0.0025 undiluted serum 



do 0.15 c.c. = 0.0015 undiluted serum 

do 0.10 c.c. — 0.0010 undiluted serum 

1-1000 dilution of the ambo- 
ceptor serum, in salt solu- 
tion 0.75 c.c. = 0.00075 undiluted serum 

do 0.5G c.c. = 0.00050 undiluted serum 

do 0.35 c.c. — 0.00035 undiluted serum 

do 0.25 c.c. = 0.00025 undiluted serum 

do 0.15 c.c. = 0.00015 undiluted serum 

do 0.10 c.c. = 0.00010 undiluted serum 



One tube containing no amboceptor is also set 
up to serve as a control. The content of all tubes 
is then brought to a like volume (2.1 to 2.5 c.c.) 
by the addition of salt solution. After being 
thoroughly mixed the tubes are placed at 37° for 
2 hours. At the end of this time one notes the 
smallest amount of amboceptor able to cause com- 
plete haemolysis. For the test proper one and 
one-half to two times this amount is employed, 



80 



SERODIAGNOSTIC METHODS. 



the amboceptor being diluted so that the required 
amount will be contained in from 0.1 to 0.2 c.c. 
of fluid. 

Titration of the Complement. — Now place in 
each of a series of test-tubes the amount of ambo- 
ceptor thus determined (i.e., 1 unit, which is the 
minimum amount required to effect lysis), 1 c.c. 
of the blood-cell suspension, and decreasing 
amounts of the complement serum. Of the last 
approximately the following amounts may be 
used: 

Complement serum undiluted . . .0.1 c.c. 
Complement serum diluted 10 

times 0.75 c.c. = 0.75 undiluted serum 

Complement serum diluted 10 

times 0.50 c.c. = 0.050 undiluted serum 

Complement serum diluted 10 

times 0.35 c.c. = 0.035 undiluted serum 

Complement serum diluted 10 

times 0.25 c.c. = 0.025 undiluted serum 

Complement serum diluted 10 

times 0.15 c.c. = 0.015 undiluted serum 

Complement serum diluted 10 

times 0.10 c.c. — 0.010 undiluted serum 

Complement serum diluted 10 

times 0.0 c.c. = 0.0 undiluted serum 



The total content of each tube is again brought 
to a like volume by the addition of salt solution, 
the contents mixed, the tubes placed in the incu- 
bator for 2 hours, and the result noted. The 
amount of complement to be used in the test is 



SEKODIAGNOSTIC METHODS. 81 



one and a half to two times the unit or smallest 
solvent dose as thus determined. 

The hemolytic system, consisting of 1 c.c. 
blood-cell suspension and the amounts as above 
fixed of amboceptor and complement, is thus estab- 
lished. It must be remembered, however, that 
the amboceptor serum employed must be of such 
a strength that the minimum effective dose is con- 
tained in from 0.001 to 0.0005 c.c. of the undiluted 
serum (1000 to 2000 units to the c.c), when 0.1 c.c. 
of guinea-pig complement is used. 

Titration of the Proteid or Bacterial Immune 
Serum. — This consists in determining in what 
dilution the serum is capable of hindering haemol- 
ysis in the above-established system when it is 
mixed with the corresponding antigen, as, for 
example, with human proteid or the typhoid bacil- 
lus extract, as the case may be. The method 
follows : 

Assuming that we are to test for human pro- 
teid in a blood-stain, the following three series of 
tests are set up : 

Series a contains 0.1 c.c. human serum diluted 
1-100. 

Series b contains 0.1 c.c. human serum diluted 
1-1000. 

Series c contains 0.1 c.c. physiological salt 
solution. 

To all tubes in each series are then added the 
6 



82 SERODIAGNOSTIC METHODS. 



proper amount of complement serum as above 
determined, and decreasing amounts of the anti- 
human immune serum, somewhat as follows: 



Immune serum, undiluted 

Immune serum, diluted 1-10 . . . 

Immune serum, diluted 1-10 . . . 

Immune serum, diluted 1-10... 

Immune serum, diluted 1-10... 

Immune serum, diluted 1-10 . . . 

Immune serum, diluted 1-10... 

Immune serum, diluted 1-10 . . . 

Immune serum, diluted 1-10... 



0.1 c.e. 

0.75 c.c. = 0.075 undiluted serum 
0.50 c.c. = 0.050 undiluted serum 
0.35 c.c. = 0.035 undiluted serum 
0.25 c.c. = 0.025 undiluted serum 
0.20 c.c. = 0.020 undiluted serum 
0.15 c.c. = 0.015 undiluted serum 
0.10 c.c. = 0.010 undiluted serum 
0.0 c.c. = 0.00 undiluted serum 



All the tubes are brought to a like volume, 
mixed and placed in the incubator for one hour at 
37°, to afford an opportunity for complement- 
fixation to take place. One c.c. of the blood-cell 
suspension, and the proper amount of amboceptor 
serum (2 units), are then added to all the tubes, 
which are then returned to the incubator for 2 
hours. Since the control series c contains anti- 
serum, but no antigen (human serum), it must 
show complete haemolysis, while the other two 
series must show more or less marked inhibition 
of haemolysis, if the antiserum is active. Since 
it is necessary, for the forensic determination of 
human blood or proteid, that the antiserum shall 
permit the recognition of the same in dilutions 
up to 1 in 10,000 (i.e., in 1-10 c.c. of a 1-1000 
dilution), such an antiserum only can be em- 



SERODIAGNOSTIC METHODS. 83 



ployed for the test as shows marked complement 
deviation (inhibition of haemolysis) in the series b. 
For the test proper one uses an amount of the 
antiserum only slightly larger than the minimum 
inhibiting dose as determined above. 

Liidke employed, in his experiments on the 
recognition of typhoid bacillus antibody in the 
blood of patients 0.1 c.c. of his bacillary extract. 
This corresponds to our series a above ; one might 
then, for the series b, use 0.01 c.c. of such an 
extract. 

The tests, somewhat elaborate, indeed, pre- 
liminary to the test proper are thus completed. 
As all the fluids employed except the complement 
can be kept almost indefinitely frozen (the freez- 
ing apparatus called "Frigo," of Morgenroth, 
and sold by the Kny-Scherer Company, of New 
York, serves admirably for this purpose), a single 
titration serves for a long time, The titration of 
the complement only needs to be repeated before 
each test, and this causes little delay. 

The Test Proper. — In each of a series of test- 
tubes place the following: 

(1) The titrated amount of complement 
serum. 

(2) The titrated amount of antihuman (or 
antibacterial) serum. 

(3) Decreasing amounts of the fluid to be 
tested. 



84 SERODIAGNOSTIC METHODS. 



For the forensic determination of blood, one 
would use about the following amounts of the 

last: o.l c.c. dilution 1-10 =0.01 c.c. 

0.1 c.c. dilution 1-100 =0.001 c.c. 
0.1 c.c. dilution 1-1000 =0.0001 c.c. 
0.1 c.c. dilution 1-10000 = 0.00001 c.c. 

0.0 c.c. = 0.0 c.c. 

In the determination of antibacterial sub- 
stances in the blood, somewhat larger amounts, 
ranging from 1 c.c. downwards to 0.001 c.c, are 
employed. Tubes without antiserum serve as 
controls. 

The tubes, having been brought to a like vol- 
ume and mixed, are placed in the incubator for 
one hour. One c.c. of the blood-cell suspension 
and 1.5 to 2 units of the corresponding ambo- 
ceptor are then added. After a second incubation 
of two hours, all the controls will show haemolysis, 
while in the test series proper a more or less 
marked inhibition of haemolysis will be observed 
if the test is positive. The method is fully as 
specific as the precipitation method, and because 
of the use of small amounts of antiserum, the 
danger of heterologous reactions is very slight. 

DETERMINATION OF ANTIBODIES. 

In the serum, cerebrospinal fluid, or tissues of 
patients (in typhoid, meningococcus, gonococcus, 
and staphylococcus infections, in pneumonia, and 
in syphilis). 

Apparatus. — The apparatus needed is the 



SERODIAGNOSTIC METHODS. 85 



same as that described on page 75, except 30 
instead of (a), the perfectly clear serum, or spinal 
fluid of the patient; and in place of {d), a solution 
of the antigen, the homologous antibody of which 
is to be determined. In the case of a bacterial 
infection, therefore, the bacterial extract de- 
scribed under (e), page 77 is employed. For the 
diagnosis of syphilis or a met a syphilitic affection 
(tabes, general paralysis, etc.) an extract of 
syphilitic organs is used. (In the first five 
methods described below, and in method 7, a beef 
blood haemolytic system can be substituted for the 
sheep blood system. According to Peters, 31 this 
has the advantage that human serum contains no 
native amboceptor for beef blood-cells, and the 
clumping and precipitation which is apt to annoy 
one in the Noguchi antihuman system does not 
occur. — Trans,) 

Diagnosis of Syphilis. 
Method of Wassermann and Meier (Berlin 
Method). — Michaelis 32 gives the following instruc- 

30 Finkelstein (Berl. klin. Woch. (1909), No. 35), describes 
special glassware for use in the complement-fixation test. This 
consists of a funnel, the stem of which, 40 mm. long by 1 mm. in- 
side diameter, is calibrated. The funnel holds 7 or 8 c.c. After 
the incubation period has elapsed, the funnel, containing the 
test, is centrifugated for a few minutes, forcing the undissolved 
red cells into the calibrated stem. The height of the column of 
red cells is used as an index of the degree of haemolysis. 

31 N. Y. Med. Jour., vol. 94 (1911), 21. 

32 Berl. klin. Woch (1907). 



86 SERODIAGNOSTIC METHODS. 



tions for preparing the antigen: The liver of a 
syphilitic fetus is preserved frozen. A few days 
before the test is to be made, a small piece is 
removed. It is first thoroughly ground in a mor- 
tar with sea sand. Five parts of physiological 
salt solution and % part 5 per cent, phenol are 
then added, and the mixture is placed in the 
shaker for several hours. It is then placed for 
4 days or longer in the ice-box (but not under 0°). 
The extract is now ready for use without filtering 
or centrifugating. 

Just before the test, small portions are cleared 
by centrifugating at high speed, and, when ready 
for use, present an opalescent appearance, but free 
of visible floating particles. This fluid contains 
the syphilis antigen. 

Levaditi dries the freshly ground organ in 
vacuo over calcium chloride and sulphuric acid, 
and pulverizes it in a sterile mortar. One part 
of the powder is rubbed up with 30 parts of salt 
solution, as required, placed in the ice-box for 
12 hours, and the clear fluid remaining after 
centrifugating is used. 

The extract must meet the following require- 
ments : 

(1) 0.2 c.c. must cause complete inhibition of 
haemolysis with 0.1 c.c. of known syphilitic serum. 

(2) 0.2 c.c. must not cause any inhibition with 
0.2 c.c. of known non-syphilitic serum. 

(3) 0.5 c.c. must cause no inhibition by itself. 



SERODIAGNOSTIC METHODS. 87 



If this occurs it is diluted, but only so far that 
requirements 1 and 2 are still complied with. 

Since many extracts deteriorate rapidly on 
standing, Seligmann and Blum 33 recommend that 
the serum of each patient should be tested not 
only with one, but with three or four extracts. 

The necessary controls are the serum or spinal 
fluid of a non-syphilitic individual and of a known 
syphilitic individual, and an extract of the liver of 
a known non-syphilitic infant. 

The titration of the amboceptor, as given on 
page 79, must precede the test proper. 

One next determines the smallest amount of 
the extract which is able to bind the complement 
without the addition of immune serum. For this 
purpose, 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 c.c. respectively 
of the extract are mixed with 0.1 c.c. complement 
serum, placed in the incubator for one hour, after 
which 1 c.c. of 5 per cent, suspension of sheep's 
cells and the requisite amount of antisheep ambo- 
ceptor are added. The content of all tubes is 
brought to 2.5 c.c. by the addition of salt solution. 
The tubes are then again placed in the incubator, 
and are inspected at the end of two hours. If the 
extract inhibits haemolysis in doses of 0.5 c.c. or 
less, it must be diluted. (See requirement 3, 
above.) In the same manner one determines at 
the same time how large an amount of the ex- 
tract is required to give complete inhibition with 

^Berl. klin. Woch. (1909), No. 24. 



88 SERODIAGNOSTIC METHODS. 



0.1 c.c. of known syphilitic serum, so as to deter- 
mine whether requirement 1 is met. If this is the 
case, the test proper may be begun. 

The method of Wassermann 34 and his col- 
laborators is as follows : The serum or spinal fluid 
of the patient is mixed with complement serum 
and liver extract, and placed in the incubator for 
one hour. One c.c. of the sheep's blood suspen- 
sion and the previously titrated amount of ambo- 
ceptor serum (see page 79) are then added, the 
volume brought to 2.5 c.c. with salt solution, and 
the tubes returned to the incubator for two hours, 
after which the result is noted. The amounts to 
be used of extract and serum or spinal fluid are 
given in the table below, which reproduces an 
experiment of Wassermann and Meier. The 
necessary controls are also shown in the table. 



Blood serum, 
inactivated, or spinal fluid 
not inactivated. 



Organ extract. 



Complement. 



Result. 



Patient's 0. 2.... 
Patient's 0. 2.... 
Patient's 0 . 2 . . . . 
Patient's 0. 4.... 
Known luetic 0.2 

Luetic 0.2 

Non-luetic 0.2... 

None , 

None 

None 

None 



Luetic 0.2 
Normal 0 . 2 

None 

None 
Luetic 0.2 
Normal 0 . 2 
Luetic 0.2 
Luetic 0.2 
Luetic 0 . 4 

None 

None 



0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 




o-co 

+» moo 




Inhibition 

Haemolysis 

Haemolysis 

Haemolysis 

Inhibition 

Haemolysis 

Haemolysis 

Haemolysis 

Haemolysis 

Haemolysis 

Haemolysis 



34 Wassermann, Neisser, Bruck, and Schucht, Zeitschr. f . Hyg., 
v °l- 45 ( 1906 ) ; Wassermann and Meier, Deutsch. med. Woch. 
(1907). 



SERODIAGNOSTIC METHODS. 89 



(Neue, Munch, med. Woch., vol. 59 (1912), 121, 
insists that larger amounts of spinal fluid than 
those specified above, up to even 1.0 c.c, should 
be used. — Trans.) 

When the reaction is positive, the phenomenon 
of complement deviation (inhibition of haemo- 
lysis) will be found only in the known luetic con- 
trol and in the patient's serum, while all the other 
controls show complete haemolysis. 

Wechselmann 35 recommends the removal by 
absorption of the." complementoids, ,, because of 
the fact that certain luetic sera react negatively 
through the phenomenon of " complementoid 
stoppage.' ' For this purpose 0.9 c.c. of the inac- 
tivated patient's serum is shaken up with 3 c.c. 
of physiological salt solution and 0.6 c.c. of an 
approximately 7 per cent, suspension of freshly 
precipitated barium sulphate. After standing for 
one hour at 37°, the mixture is clarified by cen- 
trifugation. One to 2 c.c. of the centrifugate, 
representing 0.2 to 0.4 c.c. of the serum, is used 
for the test. 

Method of Taege 36 (Breslau Method). — 
( la) Taege prepares the organ extract as follows : 
The liver of a syphilitic foetus is chopped fine or 
ground in a meat-grinder. The pulp is weighed 
and mixed in a flask with four times its weight of 

35 Zeitschr. f. Immunitiitsforch. vol. 3 (1909). 
88 Miinch. med. Woch. (1908), No. 33. 



90 SERODIAGNOSTIC METHODS. 



absolute alcohol. The flask is allowed to stand at 
room temperature for 24 hours, or is placed in the 
shaker over night. It is then filtered, the filtrate 
is placed in a shallow dish, and evaporated to a 
paste in vacuo at 40° and 60 mm. pressure of mer- 
cury. One gramme of the paste is ground to a 
fine suspension in 100 c.c. of 0.85 per cent, sodium 
chloride solution, shaken for 24 hours in the 
shaker, and preserved in the ice-box. A milky 
fluid results. Its keeping qualities may be in- 
creased by adding 0.3 per cent, phenol. 

(lb) The concentration of the extract in vacuo 
may be avoided by mixing 1 part of the fresh 
ground liver with 10 parts of absolute alcohol, 
and shaking for 24 hours with glass beads. The 
filtrate is the stock solution. 

For use, 1 part of this extract is mixed with 
3 parts of salt solution. 

(2) The patient's serum inactivated, as well as 
the known syphilitic and non-syphilitic sera, are di- 
luted with 4 parts of physiological salt solution. 

(3) The complement serum is diluted with 
9 parts of salt solution. 

(4) The amboceptor serum is preserved by 
evaporating it to a dry powder in vacuo, and seal- 
ing in small brown glass vials containing 0.3 
gramme each. Dissolve the content of one vial in 
3 c.c. of distilled water, and mix 0.1 c.c. with 60 c.c. 
of salt solution for use. 



SERODIAGNOSTIC METHODS. 91 



The hemolytic system is set up as follows: 
In each of 6 small test-tubes, numbered serially, 
is placed 2 c.c. of salt solution. Then add: 

Tube 1-1 c.c. amboceptor. 

2- 0.5 c.c. amboceptor + 0.5 c.c. salt solution. 

3- 1 c.c. amboceptor diluted 1 to 2 (1 c.c. amboceptor + 2 c.c. 

salt solution) . 

4- 1 c.c. amboceptor diluted 1 to 3 (1 c.c. amboceptor + 3 c.c. 

salt solution). 

5- 1 c.c. amboceptor diluted 1 to 5 (1 c.c. dilution 1 to 2 + 

1 c.c. salt solution). 

6- 1 c.c. amboceptor diluted 1 to 7 (1 c.c. dilution 1 to 3 + 

1 c.c. salt solution). 

One c.c. complement and 1 c.c. sheep's blood- 
cell suspension are then added to all tubes, the 
tubes shaken, and placed in the incubator. 

Tube 1 should show complete haemolysis in 
15 minutes. 

Tube 2 should show complete haemolysis in 
30 minutes. 

Tubes 3 and 4 should show complete haemolysis 
in from 30 to 45 minutes. 

Tubes 5 and 6 should show complete haemolysis 
in two hours. 

If haemolysis occurs within a shorter time, the 
amboceptor should be diluted with an equal vol- 
ume of salt solution. If it takes place more 
slowly, twice as much of the stock amboceptor 
solution should be used in making the dilution for 
use (0.2 c.c. instead of 0.1). 

To test the antigen, mix 2 c.c. with the haemo- 



92 SERODIAGNOSTIC METHODS. 



lytic system (1 c.c. each amboceptor, complement 
and blood suspension). Complete haemolysis 
must occur after incubating for one hour. 

A source of error, which may possibly disturb 
the result, lies in the fact that in rare cases a 
normal serum may cause inhibition of haemolysis 
with an otherwise trustworthy antigen. This 
error may be avoided by using an antigen only 
after one has determined that it causes no inhibi- 
tion with 10 or 12 different known normal sera. 
If the antigen is weak, and produces no marked 
inhibition with known luetic sera, special tests 
must be made to determine in what amounts it 
should be used to obtain the desired result. Fi- 
nally, it is a good plan to keep on hand as large a 
stock of known luetic sera as possible, since the 
luetic sera lose their power to react when kept 
very long. 

The test proper is made as follows : 



Tube. Complement. 

1 1 c.c. + 2 c.c. salt solution. 

2 1 c.c. + 1 c.c. antigen + 1 c.c. normal serum. 

3 1 c.c. + 1 c.c. antigen -f- 1 c.c. known luetic serum. 

4 1 c.c. + 2 c.c. antigen -f- 0 c.c. serum. 

5 1 c.c. + 1 c.c. antigen + 1 c.c. patient's serum. 

6 1 c.c. + 0.5 c.c. antigen + 0.5 c.c. patient's serum 

+ 1 c.c. salt solution. 

7 1 c.c. + 0 c.c. antigen + 2 c.c. patient's serum. 



After remaining for one hour in the incubator, 
1 c.c, amboceptor and 1 c.c. sheep's blood suspen 
sion are added to all tubes, and they are returned 



SERODIAGNOSTIC METHODS. 93 



to the incubator for another hour. Tubes 1, 2, 4, 
and 7 will then show complete solution, while 
tubes 3, 5, and perhaps 6, will show no haemolysis 
when the reaction is positive. 

Method of Hoehne 37 (Frankfurt Method) 

An alcoholic extract of the liver of a syphilitic 
fcetus or infant (1 gramme of liver to 5 c.c. of 
alcohol) is used as the antigen. The extract is 
diluted with 3 volumes of salt solution, made fresh 
for each occasion. It is used in decreasing 
amounts, as 0.75, 0.5, 0.25 c.c, to which is added 
1 c.c. of a 10 per cent, solution of the inactivated 
patient's serum, while a fourth tube contains the 
serum only, with no extract. To all tubes is then 
added 0.5 c.c. of a 20 per cent, solution of guinea- 
pig serum. The tubes are brought to a like volume 
(2.5 c.c.) by adding salt solution. One thus ob- 
tains the following scheme: 

{A) The Test Proper. — Tube 1 — 0.75 c.c. extract + 1 c.c. 
10 per cent, patient's serum + 0.5 c.c. 20 per cent, guinea-pig 
serum + 0.25 c.c. salt solution. 

Tube 2 — 0.5 c.c. extract + 1 c.c. 10 per cent, patient's serum 
+ 0.5 c.c. 20 per cent, guinea-pig serum -f- 0.5 c.c. salt solution. 

Tube 3 — 0.25 c.c. extract + 1 c.c. 10 per cent, patient's 
serum + 0.5 c.c. 20 per cent guinea-pig serum + 0.75 c.c. salt 
solution. 

Tube 4 — 0 c.c. extract + 1 c.c. 10 per cent, patient's serum 
+ 0.5 c.c. 20 per cent, guinea-pig serum + 1 c.c. salt solution. 

(B) Controls. — Tube 5 — 2 c.c. 10 per cent, patient's serum 
+ 0.5 c.c. 20 per cent, guinea-pig serum. 



3T Berl. klin. Woch. (1909), No. 38. 



94 SERODIAGNOSTIC METHODS. 



Tube 6 — 1.5 c.c. extract + 0.5 c.c. 20 per cent, guinea-pig 
serum + 0.5 c.c. salt solution. 

Tube 7 — 1 c.c. extract + 0.5 c.c. 20 per cent, guinea-pig 
serum + 1 c.c. salt solution. 

Tube 8 — 0.75 c.c. extract + 0.5 c.c. 20 per cent, guinea-pig 
serum + 1.25 c.c. salt solution. 

Tube 9 — 0.5 c.c. extract + 0.5 c.c. 20 per cent, guinea-pig serum 
+ 1.5 c.c. salt solution. 

(C) Controls. — Known luetic and known non- 
luetic sera are set up as under A. 

The tubes are then placed in the incubator at 
37° for li/4 hours, after which are added 0.5 c.c. 
of a dilution of the amboceptor, freshly prepared 
for the day, and so made that the amount men- 
tioned will contain from 2y 2 to 3 hsemolytic units 
and 1 c.c. of a 5 per cent, sheep 's blood suspension. 
The tubes are then returned to the incubator for 
two hours. 

When the reaction is positive, tubes 1, 2, and 3 
will show a more or less marked inhibition of 
haemolysis, and tubes 4 to 9 will show complete 
haemolysis. 

Simplified and Improved Method of Stern 38 
(Breslau Method) — Apparatus. — 1. Extract of 
syphilitic liver, as described on p. 90, under (lb), 
but prepared in the proportion of 1 part of liver 
to 9 parts of absolute alcohol. For use the ex- 
tract is diluted with 9 parts of salt solution. 

2. 2.5 per cent, suspension of washed sheep 
blood-cells. 



Zeitsclir. f. Immunitatsforch., vol. i 3 No. 3. 



SERODIAGNOSTIC METHODS. 95 



3. The patient's serum not inactivated. This 
serves also as complement, so that the use of 
guinea-pig serum is unnecessary. Serum sent in 
from a distance, and as much as 48 hours old, and 
even blood from a cadaver, can be tested under 
favorable conditions. 

4. Antisheep amboceptor serum. For use it is 
diluted so that 1 c.c. contains 5 to 6 units for 1 c.c. 
of the 2.5 per cent, sheep's blood-cells. 

5. Known normal and known syphilitic sera, 
in the active condition, to serve as controls. 

Technique. — Tube 1 — 0.2 c.c. patient's serum + 1 c.c. 10 per 
cent, extract. 

Tube 2 — 0.2 c.c. patient's serum + 0.5 c.c. 10 per cent, extract. 
Tube 3 — 0.2 c.c. patient's serum + 1 c.c. salt solution. 
Tube 4 — 0.2 c.c. normal serum + 1 c.c. 10 per cent, extract. 
Tube 5 — 0.2 c.c. normal serum + 0.5 c.c. 10 per cent, extract. 
Tube 6 — 0.2 c.c. normal serum + 1 c.c. salt solution. 
Tube 7 — 0.2 c.c. luetic serum + 1 c.c. 10 per cent, extract. 
Tube 8 — 0.2 c.c. luetic serum + 0.5 c.c. 10 per cent, extract. 
Tube 9 — 0.2 c.c. luetic serum + 1 c.c. salt solution. 
Tube 10 — 1 c.c. salt solution + 1 c.c. 10 per cent, extract. 

All tubes are brought to a volume of 2 c.c. by 
adding salt solution, and are placed at 37° for one 
hour. Then add to each tube 1 c.c. 2.5 per cent, 
sheep's blood suspension and 1 c.c. amboceptor 
solution, and return tubes to the incubator for 
% to y 2 hour, when it will be found that most of 
the controls already show haemolysis. The tubes 
are then allowed to stand for 1 to 1.5 hours at 
room temperature, when the final reading is taken. 

Tubes which become completely or markedly 



96 SERODIAGNOSTIC METHODS. 

haemolyzed are to be regarded as negative; mod- 
erate and delayed haemolysis, with a large sedi- 
ment of intact red cells, as positive. 

When the reaction is positive, tubes 1, 2, 7, and 
8 show inhibition ; tubes 3, 4, 5, 6, 9, and 10 show 
complete solution of the red blood-cells. 

Experience with this method, which is still 
somewhat limited, is, however, most favorable. It 
seems to be considerably more delicate than the 
original Wassermann method. Non-specific in- 
hibition has, however, been observed in cachectic 
patients. 

Simplified Method of Bauer. 39 — Apparatus. — 
(1) Fresh guinea-pig serum, diluted to 10 per 
cent, with salt solution, as complement. 

(2) Organ extract. The liver of a syphilitic 
fcetus or infant is ground up and shaken over night 
with 10 volumes of absolute alcohol. The clear 
centrifugate serves as the stock solution and keeps 
indefinitely. For use it is diluted with three parts 
of salt solution. If 1 c.c. inhibits the haemolysis 
of 1 c.c. of 5 per cent, sheep's cells, in the presence 
of 0.2 c.c. of normal human serum and 0.1 c.c. of 
complement serum, a higher dilution is to be used. 

(3) Five per cent, suspension of washed 
sheep's blood-cells. 

(4) The patient's serum, heated at 56° for 
y 2 hour. 



89 Deutsch. med. Woch. (1908), No. 16. 



SERODIAGNOSTIC METHODS. 97 



(5) Known luetic and known normal serum, 
similarly inactivated. 

The rest of the apparatus is that described 
under g-h, p. 78. Since the normal (native) ambo- 
ceptor for sheep's cells present in the human 
serum tested serves as the hemolytic agent, a 
specific immune amboceptor is not required. 

Technique. — Tube 1 — 0.2 c.c. patient's serum +1.0 c.c. ex- 
tract + 1 c.c. 10 per cent, guinea-pig serum. 

Tube 2 — 0.2 c.c. patient's serum + 1.0 c.c. salt solution + 
1 c.c. 10 per cent, guinea-pig serum. 

Tube 3 — 0.2 c.c. normal serum + 1.0 c.c. extract + 1 c.c. 10 
per cent, guinea-pig serum. 

Tube 4 — 0.2 c.c. normal serum +1.0 c.c. salt solution + 
1 c.c. 10 per cent, guinea-pig serum. 

Tube 5 — 0.2 c.c. luetic serum + 1.0 c.c. extract + 1 c.c. 10 per 
cent, guinea-pig serum. 

Tube 6 — 0.2 c.c. luetic serum + 1.0 c.c. salt solution + 1 c.c. 
10 per cent, guinea-pig serum. 

The tubes are placed in the incubator for one- 
half hour, and 1 c.c. sheep's blood suspension is 
then added. The tubes are then returned to the 
incubator for one-half to one hour. Tubes 1 and 5 
should show complete inhibition of haemolysis, 
and tubes 2, 3, 4, and 6 complete solution. Only 
complete inhibition is to be regarded as indicating 
a positive reaction. 

If considerable amounts of serum are avail- 
able, several tests should be made, using varying 
amounts of the latter. In nurslings and young 
children, and in about 10 per cent, of adults, the 

7 



98 SERODIAGNOSTIC METHODS. 



reaction miscarries, because the normal ambo- 
ceptor against sheep's blood is lacking. This de- 
fect may be corrected by adding to such sera a like 
amount of normal human serum, known to contain 
the amboceptor. As lipasmic serum cannot be 
used, it is better to draw the blood for the test 
when the patient is fasting. Numerous objections 
against Bauer's method have recently been 
offered, but future experience will have to deter- 
mine whether these are just. 

Simplified Method of Tschernoguboff. 40 — Appa- 
ratus. — 1. Antihuman hsemolytic immune serum, 
derived from the rabbit (amboceptor serum). 

2. Extract of a syphilitic liver, as antigen. 
The ground-up liver of a congenitally syphilitic 
foetus is dried and powdered. When needed, 
0.5 gramme of this powder is extracted for 12 to 
15 hours in the ice-box with 25 c.c. of 95 per cent, 
alcohol. The extract is diluted with 50 to 200 
volumes of salt solution for use. 

3. The patient's blood. This serves at once 
as the complement, red cell emulsion, and as anti- 
syphilitic antibody. It is obtained as follows: 
Prick the patient's finger, and with a pipette, or 
Zeiss blood-counting pipette for white cells, draw 
up 0.1 c.c. of blood. This is repeated four times, 
the blood obtained being each time mixed with salt 
solution or liver extract, and placed in small test- 



40 Berlin klin. Woch., (1908), No. 47. 



SERODIAGNOSTIC METHODS. 99 



tubes. In tubes 1 and 2 we have 0.1 c.c. blood + 
1.0 c.c. salt solution, and in tubes 3 and 4, 0.1 c.c. 
blood + 1.0 c.c. dilute liver extract. 

The rest of the equipment is as described 
on page 78. 

Technique. — The four tubes above mentioned 
are placed for one hour in the incubator, after 
which 0.25 c.c. amboceptor serum is added to tubes 
1 and 3, and a like amount of salt solution to 
tubes 2 and 4. The tubes are then returned to the 
incubator for two hours, and are then placed in 
the ice-box. The result is read the following 
morning. Tube 1, which contains no antigen, 
should show haemolysis, and tube 2, which contains 
no amboceptor, should show none. If this is the 
case, and if tube 3, which contains the test proper, 
shows inhibition and tube 4, which is devoid of 
amboceptor, also shows no haemolysis, the result 
is positive. 

The various methods of carrying out the com- 
plement deviation test described above have in 
part already justified themselves to an extraordi- 
nary degree. It has, however, been strikingly 
demonstrated that the reaction also takes place 
with normal liver extract, though, indeed, less 
intensely, as well as with an extract of guinea- 
pig's heart muscle ; so that, in spite of its practical 
value, the theoretic basis on which it rests has 
become doubtful. 



100 SERODIAGNOSTIC METHODS. 



Method of Landsteiner, R. Muller, and Potzl. 41 — 

According to these writers, the diagnosis of 
syphilis may be simplified as follows: Instead 
of the extract of luetic tissue, the following fluid 
is used : 1 gramme of the heart muscle of a guinea- 
pig is cut up with scissors, and then ground in a 
mortar with a small amount of quartz sand. The 
finely divided, pasty material is placed in a flask 
and thoroughly shaken with 50 c.c. of 95 per cent, 
alcohol. It is then digested for two hours at 60°, 
during which time it is occasionally shaken. It 
is then filtered through paper, and the filtrate 
kept at room temperature. 

For the rest, the same equipment is required 
as above, except that Muller uses a 50 per cent, 
emulsion of sheep's blood instead of a 5 per cent, 
emulsion. 

Technique. — In each of a series of test-tubes 
place 10 drops of salt solution and 1 drop of 
complement serum ; then add to 

Tube 1, 1 drop of the patient's serum, inacti- 
vated. 

Tube 2, 1 drop of the patient's serum, inacti- 
vated + 2 drops extract. 

Tube 3, 1 drop known luetic serum, inactivated. 

Tube 4, 1 drop known luetic serum, inactivated 
+ 2 drops extract. 



"Wien. klin. Woch., 1907, and Muller, ibid., 1908. 



SERODIAGNOSTIC METHODS. 101 



Tube 5, 1 drop known normal serum, inacti- 
vated. 

Tube 6, 1 drop known normal serum, inacti- 
vated + 2 drops extract. 
Tube 7, 2 drops extract. 

After remaining at 37° for one hour, 1 drop 
of the blood suspension and double the amount of 
amboceptor required to effect haemolysis are 
added. The tubes are returned to the incubator 
for 1% hours, when the result is read. 

Cancer Diagnosis. 

Method of von Dungern. 42 — Apparatus, — (a) 
Antigen Solution. — This is prepared in one of the 
two following ways: 1. The tumor tissue, freed 
as much as possible of fat and degenerated por- 
tions, is ground fine, and mixed with twenty vol- 
umes of acetone. Extract for one or two weeks 
at room temperature, with occasional shaking. 
Filter. For use, evaporate a portion of the ace- 
tone solution to dryness at 37°, and take up with 
half the amount of absolute alcohol. Dilute this 
with salt solution according to the results of the 
titration, which is carried out as in the case of the 
alcoholic syphilis antigens. Of three antigens, 
Von Dungern used one diluted 1-40, and the others 
diluted 1-10. The largest amount, twice which 
is not of itself anticomplementary, is to be used 



"Munch, med. Woch., vol. 59 (1912), G5, 1098, 2854. 



102 SERODIAGNOSTIC METHODS. 



for the test. 2. Eeceive 10 c.c. human blood (pref- 
erably obtained from a patient suffering from 
general paralysis) in a container in which has 
been placed 0.1 c.c. of a 20 per cent, solution of 
sodium oxalate. Wash the cells three times with 
physiological salt solution, and after the last 
washing centrifugate strongly to produce as thick 
an emulsion as possible. Weigh this and to one 
part by weight of cells, add twenty parts by vol- 
ume of C. P. acetone. Allow this to stand for 
three days at room temperature, with occasional 
shaking. Filter, evaporate the nitrate to dryness 
in the incubator, and take up the residue with 
enough 96 per cent, alcohol to form a 1 per cent, 
solution. Usually this requires about ten times as 
much alcohol as was used of the acetone. The 
extract is completely soluble in the alcohol if the 
latter is added as soon as the acetone has evapo- 
rated. For use, the alcoholic solution is slowly 
mixed with four parts of physiological salt solu- 
tion, and thoroughly shaken. 0.8 c.c. is the usual 
dose for the reaction, but this should of course 
be fixed by titration. The salt solution should 
be made with C. P. sodium chloride, Kahlbaum. 

Von Dungern prefers the extract of blood- 
cells to that made from cancer tissue. 

(b) The serum to be tested, and when available, 
positive and negative controls. Draw the blood 
from the vein aseptically and, as soon as clotting 
takes place, remove the serum and preserve in a 



SERODIAGNOSTIC METHODS. 103 



sterile container for one or two days in the ice- 
box. Then add two volumes of n/50 caustic soda 
solution and inactivate the mixture for one-half 
hour at 54° (not 56°). The n/50 caustic soda so- 
lution is prepared by diluting n/10 caustic soda 
with four volumes of physiological salt solution. 
The stock solution of caustic soda must be accu- 
rately prepared from chemically pure materials; 
it must be free from carbonate, and must be kept 
protected from contact with the air. 

(c) Fresh guinea pig serum, diluted 1-20. 1 
c.c. is used. 

(d) Five per cent, emulsion of washed beef 
(or sheep, Trans.) cells and the homologous im- 
mune serum. The unit of amboceptor is the 
amount which completely hemolyses 1 c.c. of the 
blood cells in 2 hours in the incubator, in the 
presence of 1 c.c. of the diluted complement serum. 
Use 2 units for the test. 

(e) Test-tubes, pipettes, salt solution, etc., as 
for other tests of the sort. 

Technique. — In a series of tubes place 0.6, 0.3, 
0.15, and 0.075 c.c, respectively, of the serum- 
caustic soda mixture above described. To each 
tube add 1 c.c. of the diluted complement serum 
and the proper amount of antigen, as determined 
by preliminary tests. Bring all tubes to a like 
volume, mix thoroughly, and allow to stand for 
three hours at room temperature. Then add to 
each tube 1 c.c. blood suspension and 2 units of 



104 SERODIAGNOSTIC METHODS. 



amboceptor. The result is read off after three 
hours, again at room temperature, and without 
shaking. Similar tests should be made with posi- 
tive and negative control sera, when these can be 
obtained. One control tube in each set should con- 
tain 0.2 c.c. serum and 1 c.c. complement, but no 
antigen. 

The above form of the test appeared in the 
last report above cited, under date of December 
24, 1912. With this technique Von Dungern has 
obtained over 90 per cent, positive reactions in 
known cases of cancer. Rarely syphilis and sur- 
gical tuberculosis have given positive reactions. 
Von Dungern 's experience now embraces 422 
cases. The translator obtained highly satisfac- 
tory results in a series of about 30 cases, em- 
ploying the technique recommended in the earlier 
reports above cited. Reports from other ob- 
servers are somewhat contradictory, due perhaps 
to variations in the technique employed. 

Diagnosis of Gonorrhoea (Method of Schwartz 
and McNeil). 43 — Apparatus. — (a) Antigen: Slants 
of several strains of the gonococcus (9 to 12 were 
used by S. and McK), grown for 24 to 48 hours on 
Tholman's medium containing 2 per cent, of glu- 
cose, are washed off with sterile 0.85 per cent, 
salt solution, 2 c.c. for each tube. The bacteria 



"Am. Jour. Med. Sc., vol.141 (1911), 693. 



SERODIAGNOSTIC METHODS. 105 



may be scraped from the surface of the medium 
with a platinum loop. Mix the several strains, 
pour the mixture into a sterile bottle, and heat in 
a water-bath at 56° for 30 minutes. Then either 
(1) place in a shaking apparatus and shake for 
24 hours, centrifugate, decant the supernatant 
fluid, and place the latter in a tightly corked, 
sterile bottle, add a capillary drop of lysol and 
preserve in the ice-box; or (2) allow the original 
suspension to stand in the incubator for several 
hours, at 37°, instead of shaking. Complete the 
preparation as above. 44 

The dose of the antigen to be used in the test 
is an amount which completely inhibits haemolysis 
of a fixed amount of red-cell suspension in the 
presence of a fixed amount of positive serum and 
complement, provided that twice this amount is 
not anticomplementary. 

The rest of the apparatus is identical with 
that used for the Wassermann test, page 76, 
et seq. The haemolytic system may be either a 
sheep cell system, or a human cell system, as in 
the Noguchi method. 

Technique. — The antigen is titrated against a 
known positive serum or against the antigono- 
coccic serum of Torrey, as prepared by Parke, 
Davis & Co. 



t 44 This antigen, ready for use, may be obtained from Parke, 
Davis & Co. 



106 SERODIAGNOSTIC METHODS. 



The titration of the complement and ambocep- 
tor is carried out as already described on pages 79 
and 80, using, however, 0.05 c.c. of a 5 per cent, 
suspension of sheep's cells instead of 1.0 c.c. 
For the test proper the customary excess of com- 
plement and amboceptor is employed. 

The test and necessary controls are shown in 
the following table : 



Patient's 
serum, inac- 
tivated at 
56°. 


Antigen. 


Comple- 
ment, 10 
per cent. 


Amboceptor. 


Sheep's 
cells, 5 per 
cent. 


Haemolysis 
positive case. 


0.15 c.c. 


0 


0.05 


GO -i 

(4 


0.05 


0.05 


Complete 


0.10 c.c. 


0 


0.05 


3 

O 


0.05 


0.05 


Complete 


0.05 c.c. 


0 


0.05 


CM 


0.05 


0.05 


Complete 




double 




U 






• ( 


titrated 


| 0.05 


o 


0.05 


0.05 


Complete 




quantity 




o 
CO 








0 


0 


0.05 


0.05 


0.05 


Complete 


0 


0 


0 




0.05 


0.05 


None 


0 


0 


0.05 


si 


. 0 


0.05 


None 


0.15 { 


titrated 


}0.05 




0.05 


0.05 


None 


quantity 


■§• 


0.05 


0.05 


None 


0.10 


Same 


0.05 


a 


0.05 


0.05 


None 


0.05 


Same 


0.05 


<s 

J3 


0.05 


0.05 


None 


Known 














positive 






_fl 








0.10 


Same 


0.05 


CD 

a 


0.05 


0.05 


None 


0.10 


0 


0.05 


£ J 


0.05 


0.05 


Complete 



The test is negative in the acute stages of the 
disease, but is recommended as an aid to differen- 
tial diagnosis between the chronic conditions due 
to the gonococcus and similar conditions due to 
other causes. It is not yet proved that a positive 
reaction indicates the presence of an active focus 



SERODIAGNOSTIC METHODS. 107 



of gonorrhoeal infection, but this is probably the 
case. (S. and McN.) 

Schwartz 45 states that subsequent tests have 
fully confirmed the early favorable impression of 
the test. 

Diagnosis of Tuberculosis (Method of Ham- 
mer). 40 — Apparatus. — Antigen: This is a mixture 
of old tuberculin and an extract of tuberculous 
tissue. It is important that tissue consisting of 
the tubercular lesion alone should be used so far 
as possible. Hammer prepared his extract from 
the tubercular granulations in the capsule of a 
tubercular knee-joint. It may be extracted in 
either of the two following ways: (a) Cover the 
tissue with 4 parts of alcohol and extract for three 
to five days. Filter and dilute for use with 3 parts 
of physiological salt solution. 0.4, 0.2, and 0.1 c.c. 
of this dilution are tested against 0.1 c.c. known 
tubercular serum. For the test proper use the 
largest amount, twice which amount is not in itself 
anticomplementary. Or (&), the tubercular tissue 
may be covered with 9 parts of acetone and ex- 
tracted for 10 days. Filter and evaporate to dry- 
ness at 37°. The dry residue of 10 c.c. of the 
acetone extract is dissolved in 10 c.c. of absolute 
alcohol, and diluted for use with 10 parts of 
physiological salt solution. It is titrated as above. 



45 Personal communication. 

40 Munch, med. Wochenschr., 59 (1912), 1750. 



108 SERODIAGNOSTIC METHODS. 



Mixtures of old tuberculin and extract are then 
prepared as follows: 

0.04 c.c. tuberculin + 0.4 c.c. extract. 

0.02 c.c. tuberculin + 0.2 c.c. extract. 

0.01 c.c. tuberculin + 0.1 c.c. extract. 

0.005 c.c. tuberculin + 0.05 c.c. extract. 

Of these the largest amount is used, twice 
which amount is not in itself anticomplementary. 
The extract alone and the tuberculin alone each 
reacts with a certain proportion of the cases. The 
largest percentage of positive reactions is given 
by a mixture as above. 

The other apparatus required is the same as 
that described under the von Dungern test for 
carcinoma. 

Technique. — The complement and amboceptor 
are titrated as for the von Dungern test. For the 
test proper, place in the several tubes arranged for 
the patient's serum and the customary controls 

1 c.c. of the diluted complement. To the front 
row add the titrated amount of antigen. Mix thor- 
oughly, and then add 1-10 c.c. of the several sera 
inactivated for half an hour at 56°. This mixture 
is allowed to stand for three hours at room tem- 
perature, after which 1 c.c. of 5 per cent, suspen- 
sion of beef (or sheep, Translator) cells and 

2 units of the corresponding amboceptor are 
added. The reading is made after one hour in 
the incubator and when all the controls are com- 
pletely dissolved. 



SERODIAGNOSTIC METHODS. 109 



Hammer 's article is merely a preliminary con- 
tribution. His series consisted of 46 cases of 
known tuberculosis, of which 97.8 per cent, gave 
a positive result. The one case which failed to 
react was possibly cured. 

Cobea Venom Reactions. 

Inhibition of Cobra Venom Haemolysis (Psycho- 
reaction of Much and Holzmann). 47 — Principle. — In 
many psychoses the serum of the patient has the 
ability to neutralize the hemolytic power of cobra 
toxin. The clinical significance of the reaction 
has not yet been determined. 

Apparatus. — (a) A solution of cobra venom. 
Dissolve 0.2 gramme toxin in 10 c.c. of distilled 
water and add 10 c.c. of glycerin. Keep this stock 
solution in the ice-box. 

(b) A freshly prepared 10 per cent, suspen- 
sion of washed human red blood-corpuscles (de- 
fibrinated placental blood). 

(c) The patient's serum, inactivated by heat- 
ing for one-half hour at 56°. 1.5 to 2 c.c. are 
required. 

(d) Small test-tubes, 6 or 7 mm. in diameter. 

(e) Pipettes, 1 c.c. in 1-10's. 
(/) Physiological salt solution. 
(g) An incubator, at 37°. 

47 Munch, med. Woch. (1909), No. 20, and Omorokow, Berl. 
klin. Woch. (1909), No. 41. 



110 SERODIAGNOSTIC METHODS. 



Technique. — The venom solution for the test is 
prepared by diluting the stock solution with 50 
volumes of salt solution, e.g., 0.2 c.c. toxin to 
9.8 c.c. salt solution. In each of a series of tubes 
place 0.35 c.c. patient's serum and decreasing 
amounts of venom, 1.0, 0.5, 0.25, and 0.1, respec- 
tively. Then add to each tube 0.5 c.c. of the blood- 
cell suspension. The total content of all tubes is 
then made 1.85 c.c. by the addition of salt solu- 
tion. A like series of tubes, with the patient's 
serum omitted, serves as a control. One thus 
obtains : 



Tube. Serum. Cobra toxin. Blood. Salt solution. 

1 0.35 1.0 0.5 0 

2 0.35 0.5 0.5 0.5 

3 0.35 0.25 0.5 0.75 

4 0.35 0.1 0.5 0.9 

5 0 1.0 0.5 0.35 

6 0 0.5 0.5 0.85 

7 0 0.25 0.5 1.1 

8 0 0.1 0.5 1.25 



Mix thoroughly, and place the tubes in the 
incubator for two hours, then in the ice-box for 
22 hours more. Before the final inspection, all 
tubes should be shaken thoroughly. When the 
reaction is positive, the tube containing the 
patient's serum, and the smallest amount of toxin 
able to produce haemolysis in the absence of serum, 
remains non-transparent, i.e., shows inhibition of 
haemolysis. In the above table, for example, if 
solution occurs in tubes 5, 6, and 7, but is absent 



SERODIAGNOSTIC METHODS. Ill 



in 8, the minimum dissolving dose of toxin is 0.25, 
and tube 3, which also contains this amount of 
toxin, must show inhibition. 

Cobra Venom Reaction of Weil. 48 — Principle. — 
Human blood-cells show varying degrees of re- 
sistance to the haemolyzing power of cobra venom. 
In syphilis this resistance is increased more than 
in any other condition. In the earliest stages of 
the disease the cells may be hypersensitive. 

Apparatus. — (1) A solution of cobra venom. 
The stock solution is a 0.5 per cent, solution, made 
very accurately with 0.9 per cent, salt solution. 
It deteriorates rapidly unless kept frozen. For 
use, dilutions of 1-10,000, 1-20,000, 1-30,000, and 
1-40,000 are prepared and kept constantly at a 
low temperature. 

(2) The blood-cells to be tested. Draw the 
blood from one of the veins at the elbow and dis- 
charge about 2 c.c. into 2 per cent, sodium citrate 
in 0.9 per cent, sodium chloride. The latter must 
be prepared fresh, or, at most, the day before, 
and kept on ice. The mixture of blood and citrate 
solution must not be shaken. The cells must be 
washed at least four times in 0.9 per cent, salt 
solution. The washing is done in accurately cali- 
brated tubes, and the last washing always at the 
same speed and for the same length of time. Ac- 

^Infec. Dis., vol. 6 (1909), 688, and Schwartz, K Y. Med. J., 
95 (1912), 23. 



112 SERODIAGNOSTIC METHODS. 



curate dilution of the red cells is very important. 
The cells are then made up to a 4 per cent, sus- 
pension with salt solution, which has been kept 
on ice if the cells are to be tested the same day. 
The suspension is to be placed on ice for a short 
time before the test is made. 

Technique. — As the dry venom does not vary, 
a single preliminary titration against known nor- 
mal and known syphilitic serum serves for all. 

Mix 1 c.c. of the cells with 1 c.c. of each of the 
venom dilutions. Incubate at 37° for one hour. 
Mix thoroughly, and place in the ice-box over 
night. In the morning again mix thoroughly, and 
make the final reading an hour later. The venom 
used by Weil titrated as follows: 

Non-haemolysis at 1-10,000 = strongly positive. 
Moderate haemolysis at 1-20,000 = positive. 
Partial haemolysis at 1-30,000 = negative. 
Complete haemolysis at 1-40,000 = hypersensitiveness. 

Fresh blood-clot may be broken up in the 
citrate solution, prepared as above, and tested, 
using the venom twice as strong. There are three 
criteria which aid in determining the result, viz., 
the rapidity with which the haemolysis begins, the 
degree of completeness at the time of the final 
reading, and the tendency of the red cells to sink 
rapidly to the bottom. These conditions are all 
reversed in positive tests. 

The method is said to have the following 
advantages over the Wassermann method: It is 



SERODIAGNOSTIC METHODS. 113 



simpler and less laborious; a smaller amount of 
blood is required; scarlatina and leprosy do not 
react positively; jaundice does not interfere with 
the test ; it is more sensitive than the Wassermann 
in the late, untreated, and dormant cases in which 
diagnosis is especially apt to be difficult ; it is less 
quickly abolished by treatment, affording a fur- 
ther diagnostic and therapeutic test. 

Cobra Venom Reaction of Kraus, Graff, and 
Ranzi. 49 — Principle. — In certain conditions human 
blood-serum possesses the ability to activate the 
haemolysis of horse's red blood-cells by cobra 
venom. 

Applications. — About 70 per cent, of the can- 
cer sera react positively, and the reaction may 
remain positive for years after operation, in 
the absence of any indication of recurrence. It is 
invariably strongly positive in pregnancy, begin- 
ning with the fourth month, and disappearing 
about three weeks after parturition. Icterus and 
advanced tuberculosis may also react positively. 
(Graff and Zubrzycki. 50 ) 

Apparatus. — (a) Solution of cobra venom. 
The stock solution is 1 per cent, in equal parts of 
glycerin and distilled water. It should stand sev- 
eral days before it is used, and keeps extraordi- 



Wien. klin. Woch. (1911), No. 28. 
Munch, med. Woch., vol. 59 (1912), 574. 

8 



114 SERODIAGNOSTIC METHODS. 



narily well in the ice-box. A dilution of 1-5000 
is made from this just before use. 

(b) Patient's serum. Inactivate for 20 min- 
utes at 50°. 

(c) Horse's red blood-cells, washed three or 
four times with salt solution and made up to a 
10 per cent, suspension in salt solution. Blood a 
week old can be used after recentrifugating to 
remove cells spontaneously haemolyzed. 

(d) Test-tubes of the ordinary size. 

(e) Salt solution. 

(/) Volumetric pipettes, 1 c.c. in 1-100 's and 
10 c.c. in 1-10 

(g) Incubator at 37°. 

Technique. — In two series of four tubes each, 
place 0.1, 0.2, 0.3, and 0.5 c.c. of the inactivated 
patient's serum, and fill all tubes to 0.5 c.c. To 
the first series add 0.3 c.c. of the cobra venom solu- 
tion, 1-5000, and to the second series 0.15 of the 
same. Finally, add to all tubes 5 drops of the 
10 per cent, horse blood suspension. Shake vigor- 
ously, and place in the incubator at 37°. Inspect 
at the end of 15 and 30 minutes, and after one, 
two, and three hours. The final inspection deter- 
mines the result, which Graff and Zubrzycki indi- 
cate as follows: 

& = no solution, + = trace of solution, + + = 
partial solution, + + + = complete or almost 
complete solution. 



SERODIAGNOSTIC METHODS. 115 



Antifekmentative Beactions. 
estimation of the antitkypsin content of the 

BLOOD. 

Principle. — One determines what amount of 
trypsin is neutralized, as to its digestive power, 
by a certain amount of serum or blood. The sub- 
stance to be digested may be either Loeffler's 
blood-serum, as ordinarily used by bacteriologists, 
or a solution of casein. In the former case the 
criterion for the occurrence of digestion is the 
formation of a dimple on the surface of the serum ; 
in the latter, the non-appearance of a cloud, due 
to the precipitation of casein, on the addition of 
acetic acid. 

Practical Application. — (The antitrypsin of 
the blood is most markedly increased in cancer. 
The method has been used chiefly for cancer diag- 
nosis. — Trans.) 

Method of Marcus 51 (Modification of the 
Method of Muller and Jochmann) 52 . — Appara- 
tus. — (1) Trypsin solution: Thoroughly shake in a 
test-tube 0.1 Gm. trypsin (Kahlbaum) with a mix- 
ture consisting of 5 c.c. undiluted glycerine and 
5 c.c. distilled water. Then place at 55° for one- 
half hour, and, after a second thorough shaking, 
filter. The solution keeps a long time. 



"Berlin, klin. Woch. (1908), No. 14, and (1909), No. 4. 
52 Munch, med. Woch. (1909), Nos. 29 and 31. 



116 SERODIAGNOSTIC METHODS. 



(2) Loeffler's blood-serum, 11 or 12 dishes. 
The mixture of 3 parts sterile beef or sheep's 
serum and 1 part bouillon, made up to contain 
1 per cent, peptone, 0.5 per cent, sodium chloride, 
and 1 per cent, dextrose, is poured into sterile 
Petri dishes, and solidified at 90° to 95° C. The 
plates cannot be used immediately after they are 
prepared, but must stand for several days at 
room temperature till they have lost their water 
of condensation and have acquired a firm, elastic 
surface. 

(3) Blood or serum of the patient, obtained 
three or four hours after the last meal. A few 
drops suffice. 

(4) An incubator at 37°. 

(5) Small watch-glasses or hollow ground 
slides in which to receive the blood, and to pre- 
pare the mixture of serum and trypsin. 

(6) A platinum loop. 

Technique. — Serum or whole blood may be 
used equally well for the test. If whole blood is 
used, a couple of drops are received on a hollow 
ground slide, and may be applied immediately 
to the Loeffler serum. For this purpose one pre- 
pares various mixtures of trypsin solution and of 
serum or blood. For example, 1 loop of serum or 
blood with i/ 2 , 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 loops 
of the trypsin solution. From each of these mix- 
tures, which are prepared in watch-glasses or hol- 
low slides, 6 or 8 loops are placed on the surface 



SERODIAGNOSTIC METHODS. 117 



of the serum in such a way that they will not 
become confluent. One Petri dish receives trypsin 
solution only, without blood or serum. 

All plates are then placed in the incubator, 
where they remain for five hours. At the end of 
this time there will be found, wherever the trypsin 
has not been completely paralyzed by the blood, 
shallow dimples on the surface of the Loeffler 
medium. If, for example, dimple formation 
occurs with mixtures containing 1 part blood to 
3 parts trypsin, but is lacking with mixtures con- 
taining 1 part blood to 2 parts trypsin, the blood 
is said to have a titre of 3:1. By comparing the 
titre thus obtained with that of normal blood, 
one determines whether in the given case the 
antitryptic power of the blood is normal, de- 
creased, or increased. (The greater the amount 
of trypsin that is required to cause dimple for- 
mation, the higher the titre, or, in other words, 
the greater is the antitryptic power of the blood.) 

Method of Bergmann and Meyer. 53 — Appa- 
ratus. — (1) A 1 per mille solution of trypsin sice. 
(Gruebler) : dissolve 0.5 Gm. trypsin in 50 c.c. 
physiological salt solution, add 0.5 c.c. normal soda 
solution, and make up to 500 c.c. 

(2) Casein solution: 1 Gm. casein (Ehenania) 
is dissolved in 100 c.c. n/10 NaOH solution, with 



Berl. klin. Woch. (1908), No. 37. 



118 SBRODIAGNOSTIC METHODS. 



the aid of gentle heat. Neutralize to litmus with 
n/10 HC1 solution and make up to 500 c.c. 

(3) Acetic acid solution: 5 c.c. acetic acid, 
45 c.c. alcohol, and 50 c.c. water. 

(4) The patient's serum, diluted 20 times with 
salt solution. 0.1 to 0.2 c.c. suffices. 

(5) Small test-tubes. 

(6) Pipettes, 2 c.c. in 1-10 's. 

Technique. — (a) Titration of the trypsin solu- 
tion. This must precede the test proper: In each 
of several test-tubes place 2 c.c. of the casein 
solution and decreasing amounts of the trypsin 
solution, as, for example, 1.0, 0.9, 0.8, 0.7, . . . 
0.2. Shake carefully and place in the incubator 
for one-half hour. Then add the acetic acid solu- 
tion drop by drop, observing which tube shows 
cloudiness after a few minutes. The tube contain- 
ing the smallest amount of trypsin, and which re- 
mains perfectly clear, contains the "completely 
digesting dose," which is used for the test proper. 

(b) Estimation of the antitrypsin: In each 
of 6 tubes place 0.2 c.c. of the patient's serum, and 
increasing amounts of the trypsin solution, begin- 
ning with the completely digesting dose, and in- 
creasing by 0.1 c.c. Add 2 c.c. casein solution to 
each tube and bring all the tubes to a like volume. 
Incubate as before for one-half hour at 37°. Acid- 
ify as before and again note the tube containing 
the smallest amount of trypsin in which cloudiness 



SERODIAGNOSTIC METHODS. 119 



can be seen. The amount of trypsin paralyzed by 
the antitrypsin of the serum is thus determined. 
If, for example, the completely digesting dose is 
0.7 c.c, and if, in the presence of serum, 2.0 c.c. 
of trypsin are required to bring about complete 
digestion, then : 

2.0 c.c. — 0.7 c.c. = 1.3 c.c, 

or the amount of trypsin paralyzed by the amount 
of serum used (in this case 0.2 c.c. of a 5 per cent, 
dilution, or 0.01 c.c. whole serum). One c.c. of 
serum, therefore, paralyzes 130 c.c. of the trypsin 
solution. Fiirst 54 expresses the result as follows : 

37° 

Antitrypsin = 130. 

hour 

Comparison of the values thus obtained with 
those given by normal serum shows whether the 
antitryptic power of the serum tested is altered. 

The Meiostagmin Eeaction. 

Method of Ascoli and Izar. 55 — Principle. — 
When the suitably diluted blood-serum of patients 
suffering with certain diseases is mixed with the 
homologous antigen and heated, there occurs a 

5 *Berl. klin. Woch. (1909), No. 2. 

55 Ascoli, Ascoli and Izar, and Izar, Munch, med. Woch., vol. 
57 (1910), pp. 62, 182, 392, 403, 842, 1170, 2129. 



120 SERODIAGNOSTIC METHODS. 



specific lowering of the surface tension of the 
serum, as measured by the stalagmometer of 
Traube. 

Practical Applications. — The method has been 
used for the diagnosis of typhoid fever, tubercu- 
losis, syphilis, ankylostomiasis, echinococcus dis- 
ease, and cancer. 

Apparatus. — (1) A solution of the homologous 
antigen. For typhoid, a solution of the products 
of autolysis of the typhoid bacillus is employed, 
prepared according to the method of Neisser and 
Shiga, 56 except that the heating to 60° is omitted. 
Wash off a 24-hour agar culture of the typhoid 
bacillus with 10 c.c. sterile physiological salt solu- 
tion, cover the emulsion with toluol, and shake 
vigorously. Place in the incubator at 37° for 48 
hours, and then filter through a germ-proof filter. 
The filtrate may be used as antigen ; or, better, an 
alcoholic extract may be prepared in one or other 
of the two following ways: (a) Dry 5 c.c. of the 
filtrate over a water-bath at 50°. To the dry resi- 
due add 10 c.c. of absolute alcohol, shake thor- 
oughly, and centrifugate. To the residue add 
10 c.c. of absolute alcohol, shake, and again cen- 
trifugate. Combine the centrifugates, and evap- 
orate on the water-bath at 50° to 5 c.c. Or (b), 
to 5 c.c. of the original aqueous filtrate add 20 c.c. 



58 Deutsch. med. Woch., vol. 29 (1903), 61. 



SERODIAGNOSTIC METHODS. 121 



of absolute alcohol. Allow the mixture to stand 
for one-half hour, centrifugate, and mix the sedi- 
ment with a fresh portion of absolute alcohol, 
10 c.c, shake thoroughly, and again centrifugate. 
Combine the two extracts, and concentrate on the 
water-bath to 5 c.c. 

The syphilis antigen is prepared by extracting 
0.5 gramme of a dried and powdered syphilitic 
spleen with 50 c.c. of absolute alcohol for two 
hours at 37°, with frequent shaking. Filter, and 
concentrate to 10 c.c. 

To prepare the tuberculosis antigen, proceed 
as follows: Rub up a (bouillon or agar) culture 
of the tubercle bacillus in a mortar, and digest for 
several days at 37° with 95 per cent, alcohol, re- 
peatedly renewed till the alcohol remains clear. 
Dry the bacillary residue at 47°, and extract with 
warm ether. Dry, and again extract with 95 per 
cent, alcohol, renewing the latter as above till it 
remains clear. Combine the alcoholic extracts, 
filter, and evaporate to dryness at 47°. Now 
pour the ether extract into the same evapo- 
rating dish which contains the residue of the 
alcoholic extract, and evaporate to dryness at 
30°. Dissolve the entire residue in absolute alco- 
hol, filter, and evaporate at 47° till a sediment 
begins to form, and again filter. Now add ether, 
drop by drop, till (having added about 1-10 the 
volume) a precipitate forms. Filter through thick 



122 SERODIAGNOSTIC METHODS. 



filter paper (Dreverhoff" No. 41), drive off the 
ether, and repeat this process till no precipitate 
forms on the addition of ether. Again evaporate 
to dryness, dissolve in absolute alcohol, and con- 
centrate to the saturation point. 

To prepare the cancer antigen, the undegener- 
ated portions of the tumor are ground in a meat- 
chopper, spread in a thin layer on glass plates, 
and dried in the incubator or in vacuo. Powder 
the dry residue in a mortar, and extract for 24 
hours at 50° with methyl alcohol, using 25 c.c. 
for each 5 grammes of tumor tissue. The flask 
should be shaken occasionally. Filter hot, allow 
the filtrate to cool to room temperature, and again 
filter through S. & S. No. 590. Dry the residue, 
and repeat the process, after which it is again 
dried and again extracted with methyl spirit. 
Again dry, and extract with warm ether, renewed 
several times in the course of 24 hours. Dry, and 
repeat the extraction with methyl alcohol several 
times till the alcohol remains colorless. Evapo- 
rate the alcoholic and ethereal extracts at 50° and 
at 37°, respectively. A yellowish-red, sticky mass 
results. Dissolve this in a large amount of water- 
free ether. Filter, and evaporate at room tem- 
perature till a powdery precipitate, slight in 
amount, forms. This solution constitutes the 
stock antigen, which, however, may have to be still 
further concentrated. Too dilute a solution keeps 



SERODIAGNOSTIC METHODS. 123 



poorly, while a too concentrated solution emulsi- 
fies badly. 

Kohler and Luger 57 obtained a more stable 
and equally effective cancer antigen as follows: 
Grind up some lecithin (ovolecithin Merck, lecithin 
Agfa, or lecithin Eichter), and extract it for 24 
hours at 50° with acetone. Filter clear through 
S. & S. filter paper No. 590. Just before being 
used it is diluted with water in such a manner that 
1 c.c. contains the largest amount which does not 
cause a marked reduction of surface tension in 
normal serum. As a rule, this dilution will be 
found to lie between 1-60 and 1-100. 

K. and L. place the normal variation at from 
three to eight fractions. 

A reaction is positive when the increase ranges 
from 24 fractions of a drop (1 drop contained 26 
fractions with the apparatus used) to 1.5 drops. 

The ankylo stoma antigen is prepared by rub- 
bing up the worms obtained from feces in a mor- 
tar, and extracting to exhaustion with 95 per cent, 
alcohol at 37°. The residue is dried, powdered, 
and again extracted. The extracts are combined, 
dried, and dissolved in absolute alcohol, and 
filtered. 

The fluid contents of an echinococcus cyst may 
be used as an antigen for the diagnosis of this 



"Wien. klin. Woch., vol. 25 (1912), 1114. 



124 SERODIAGNOSTIC METHODS. 



condition, but this gives less clearly defined results 
than an antigen prepared from the cyst wall and 
contents, according to the method given for the 
ankylostoma. 

All these stock solutions keep well, but not in- 
definitely. They should be kept protected from 
light, but not necessarily in the ice-box. Shaking 
seems to injure them. Whenever portions are 
removed for use, an absolutely dry pipette should 
be employed. 

(2) Traube's stalagmometer. Ascoli and Izar 
use the size giving 56.15 drops of distilled water at 
20°. It can be obtained from the Kny-Scherer 
Co., New York. 

(3) Serum of the patient, and of known posi- 
tive and negative controls. 

(4) Test-tubes of the standard size. 

(5) Physiological salt solution and distilled 
water. 

(6) Volumetric pipettes, 1 c.c. in 1-100 's and 
10 c.c. in 1-10 's. 

(7) Incubator at 37°. 

Technique. — The first step is the mastery of 
the instrument. One should practise until suc- 
cessive readings with the same fluid can be ob- 
tained which do not vary more than one or two 
fractions of a drop. The cleaning of the appa- 
ratus is important. It should be washed with dis- 
tilled water, then 10 per cent, caustic soda solution, 



SERODIAGNOSTIC METHODS. 125 



then with distilled water, alcohol, and finally ether. 
Just before it is used it is again washed with dis- 
tilled water, and finally rinsed twice with the fluid 
to be tested. The lower end of the instrument, on 
which the drop forms, must not be touched with 
the fingers, and care must be taken to see that 
the drop forms on the whole of this surface. 
(When in use, the instrument should be mounted 
in a clamp or holder, and jarring from heavy foot- 
steps, closing of doors, etc., must be carefully 
avoided. — Trans. ) 

The standardization of the antigen follows: 
The stock solution is diluted in varying propor- 
tions with 0.85 per cent, salt solution or with dis- 
tilled water. For the test proper, employ the 
greatest concentration of the antigen which does 
not cause an increase of more than one drop, or 
only slightly more than one drop, when mixed 
with suitably diluted normal serum and incubated 
for two hours at 37°. The test may be carried out 
somewhat as follows: With a dry pipette place 
0.05 c.c, 0.1 c.c, and 0.2 c.c. of the stock antigen 
at the bottom of dry test-tubes, add rapidly to 
each 10 c.c. of distilled water and shake vigorously. 
Now place in each of four test-tubes 9 c.c. of nor- 
mal blood-serum, diluted 1-20 with salt solution. 
The blood-serum may be diluted in other propor- 
tions, even as high as 1-100, if only a small quan- 
tity is available. To one tube add 1 c.c. of distilled 
water, and to the others 1 c.c. of one of the three 



126 SEEODIAGNOSTIC METHODS. 



antigen dilutions, and shake all tubes thoroughly. 
Count the number of drops at once, and place in 
the incubator in open tubes at 37° for two hours, 
or in a water-bath at 50° for one hour. When a 
water-bath at 50° is used, distilled water must be 
added afterward to make up the loss by evapora- 
tion. Allow all tubes to cool to room temperature, 
and repeat the count. The beginner at least 
should make all counts in duplicate. 58 Two ex- 
periments condensed from Ascoli and Izar, on 
typhoid and tuberculosis serum respectively, are 
reproduced below : 



TYPHOID SERUM. 



Patient's serum 1-10. 


Before 


After 


Normal se 
Before 


rum 1-10. 

After 


Incubation. 


Incubation. 


9 c.c.+l c.c. salt solution 

9 c.c. + l c.c. antigen 1-1000. . . . 
9 c.c.+l c.c. antigen 1-10,000. . . 
9 c.c.+l c.c. antigen 1-100,000. . 


(57 
157+1 
/58 
158 + 1 
/58+2 
158 + 1 
J 58 
157+9 


58 

68 

61+2 
61+1 
60 
60 

59+1 
59+2 


57+1 
57+2 
57+1 
57 

57+1 
57 + 1 
57 + 1 
57 


58 

58+1 

58+2 

58 

58 

58 

58 

57+9 



The optimum dilution is 1-1000 or less. A 
fresh series of dilutions can be made to determine 
this. The table also illustrates a positive reaction. 



68 An electric device, which rings a bell at the fiftieth drop, 
can be obtained if desired. 



SERODIAGNOSTIC METHODS. 127 



TUBERCULOSIS SERUM. 



Patient's serum 1-20. 


Before 


After 


Normal se 
Before 


rum 1-20. 
After 


Incubation. 


Incubation. 


9 c.c.+l c.c. antigen 1-20 


57+4 
59 

57+5 
57+3 


58-2 
59+5 
60-2 
60-5 


58+3 
59+5 
58+4 
58+2 


58+5 
60-2 
58+5 
58+4 


9 c.c.+l c.c. antigen 1-90 


59+1 

59 

59-4 
58+1 
58-3 
57+5 


60-4 
60-2 
60-1 
60-1 
60-3 
60-1 


59+5 
60-3 
59+1 
58+4 
58+5 
58+3 


60+1 
60+2 
60-4 
59+1 
59-3 
58+4 



Although the lower dilution, 1-50 to 1-70, did 
not cause an excessive increase in the normal 
serum, it failed to show an adequate increase in 
the known positive serum. The dilution selected 
for use was, therefore, one between 1-80 and 
1-100. The figures after the plus and minus signs 
in the tables indicate fractions of a drop as indi- 
cated by the calibration on the stem of the instru- 
ment. The number of such fractions to a drop 
will, of course, vary with the size of a drop. 

The test proper is carried out in essentially the 
same manner as is the preliminary titration of the 
antigen. Dilute 1 c.c. of the patient's serum (Izar 
has obtained satisfactory results with 15 or 20 
drops of whole blood diluted with distilled water, 



128 SERODIAGNOSTIC METHODS. 



and also with blood-clots dried on glazed paper, 
redissolved in distilled water and filtered through 
cotton) of known normal serum and, when obtain- 
able, of known positive serum with 19 c.c. of 0.85 
per cent, salt solution. To 9 c.c. of each dilution 
add 1 c.c. of the antigen dilution as previously 
titrated, and of distilled water, respectively. 
Count the drops at once, and again after incuba- 
tion. An increase amounting to two drops or more 
constitutes, a positive reaction. The increase in 
tuberculosis is usually less than in any of the 
other conditions in which the test has been used. 
In cancer an increase amounting to four to six 
drops often occurs. 

An extensive literature has accumulated, and 
the value of the test in diagnosis has been abun- 
dantly confirmed. The method is, however, ex- 
tremely susceptible to errors arising from faulty 
technique, and, unless all the precautions indicated 
are observed rigidly, the results are worthless. 

Phagocytic Reactions. 

estimation of the opsonic index of a serum 
according to the method of wright. 

Method of Bine and Lissner. 59 — Principle. — 
When leucocytes are mixed with bacteria and an 
opsonizing serum, phagocytosis takes place, the 



Munch, med. Woch. (1907). 



SERODIAGNOSTIC METHODS. 129 



degree of which can be estimated by counting the 
number of bacteria taken up. 

Apparatus. — (a) The patient's serum. Wright 
obtains the serum by means of a small glass tube 

Fig. 6. 




-Point- to break 



l~ . .^.pointto break, 

of the form shown in Fig. 6. After swinging the 
left arm vigorously, the proximal phalanx of the 
thumb is quickly bound with a small bandage. 
Break off both ends of the glass tube, and draw up 
the blood through the end A. Seal the opposite 
end B first, scrupulously avoiding heating the 
blood. When the cooling of the sealed end has 
drawn the blood away from the end A, the latter 
can also be sealed. After the blood has coagu- 
lated, the tube can be hung by its bent end on the 
rim of a centrifuge tube and centrifugated to com- 
plete the separation of the clot. The bent end is 

9 



130 SERODIAGNOSTIC METHODS. 



then cut off, and the serum withdrawn with a 
capillary pipette. 

The serum can, of course, be obtained in any 
other convenient manner. 

(b) The serum of normal persons, similarly 
obtained, to serve as controls. 

(c) Washed blood-cells. Place a few drops of 
blood from a normal person in a clean test-tube, 
two-thirds filled with a freshly prepared 1.5 per 
cent, sodium citrate solution. Mix thoroughly, but 
without shaking, and centrifugate. Pipette off the 
clear fluid, and replace with 0.85 per cent, sodium 
chloride, and again centrifugate. Eemove nearly 
all of the salt solution, and mix the blood-cells with 
the remainder. This constitutes the blood suspen- 
sion ready for use. The cells change after stand- 
ing only a few hours, so that a fresh suspension 
must be prepared for each test. 

(d) An emulsion of the species of bacteria 
against which the blood is to be tested. 

The method of preparing this emulsion varies 
with the species to be used. For tests on tuber- 
culosis it is recommended to use the dry, dead bac- 
teria such as are obtainable from the Hoechst 
Farbwerke. Grind up a small quantity of this 
preparation to a fine powder in an agate mortar, 
and make up to a thick emulsion by adding 1.5 per 
cent, sodium chloride solution, drop by drop. 
Sterilize by heating for one hour at 60°. It is most 
easily preserved in a glass tube, the lower end of 



SERODIAGNOSTIC METHODS. 131 



which has been drawn ont thin. At the best, it 
keeps for about 10 days. A few coarse clumps of 
bacteria are apt to form at the thin, lower end of 
the tube, and these can be removed by cutting off 
the end of the tube and resealing. 

In the case of other species, one loop of a 
12- to 24-hour agar culture is usually employed, 
rubbed up with a little physiological salt solution, 
and then diluted with more of the same fluid. 
Strubell 60 recommends, in the case of the colon 
group and the Gram-negative cocci, cultures only 
four to ten hours old. The diluting fluid for the 
tubercle bacillus and the Gram-negative cocci 
should be a 1.5 per cent, solution of sodium chlo- 
ride. Emulsions of streptococcus have to be 
shaken very vigorously to render them homo- 
geneous. To remove chains or threads remaining 
in the emulsion, the latter may be centrifugated a 
short time, and the upper part carefully poured 
from the sediment. 

Before it is used, the emulsion must be tested 
to determine whether it contains a sufficient num- 
ber of bacteria. According to Wright, the number 
should be about 7000 to 10,000 million in 1 c.c. 
The number can be readily estimated by mixing 
together equal volumes of blood and emulsion, 
smearing a drop of the mixture on a slide, fixing, 
and staining. Since 1 c.c. of blood contains about 
5000 million red cells, a field should average two 

^Deutsch. med. Woch. (1908), No. 19. 



132 SERODIAGNOSTIC METHODS. 



bacteria to one red cell. If the number is greater 
than this, the emulsion should be diluted 
accordingly. ? 

0.5 cm 



Serum\ 



leucocytes ' % 



* Airspace* 



> Air space? 
Mark 



(e) Capillary tubes made by drawing out glass 
tubing of about 5 mm. inside diameter over the 
gas flame. The capillary part should be 16 cm. 
long (Fig. 7). The upper wide end is provided 
with a rubber nipple. 



SERODIAGNOSTIC METHODS. 133 



(/) Slides, thoroughly cleaned, sterilized, and 
slightly roughened by rubbing with fine emery 
paper. Cover-glasses. 

(g) A so-called spreader, that is, a slide with 
the edge ground out slightly concave, to use in 
making the smear. 

(h) Saturated solution of mercury bichloride 
for use as a fixative. 

(i) Ziehl-Nielson carbofuchsin or carbo- 
thionin for the tubercle bacillus, eosin methy- 
lene blue, May-Griinwald (or Wright's, Trans.), 
etc. 

(j) Microscope with homogeneous immersion 
lens. 

(k) An incubator at 37°. 

(I) A Bunse.u burner. 

(m) A glass knife or file. 

Technique. — Make a mark 2 or 3 cm. from the 
end of the capillary tube. With the rubber nipple 
draw up the serum to this mark; then admit a 
bubble of air; then the blood suspension to the 
mark ; then another bubble ; then the bacterial sus- 
pension to the mark. The capillary now contains 
equal volumes of the three fluids, separated from 
one another by little bubbles of air. (Fig. 7.) 
The capillary is immediately emptied on a slide, 
or, perhaps better, on the bottom of a short test- 
tube, the fluids mixed as quickly and thoroughly 
as possible, and again drawn into the capillary 
and the end sealed. Heating of the contents must, 



134 SERODIAGNOSTIC METHODS. 



of course, be avoided. The same treatment is 
given the control tube containing the normal 
serum. 

Now place the sealed tubes for 20 to 30 min- 
utes in the incubator. After incubation, cut off 
the end of the tubes and smear the contents of 
each on a slide, using the "spreader," and avoid- 
ing the use of any pressure. Saathoff prefers to 
place the contents of the tubes on slides, and to 
mix thoroughly with a platinum loop. 

After the slides become air-dry, fix for two or 
three minutes in the sublimate solution, and then 
stain. 

One next proceeds to count the number of bac- 
teria contained in each leucocyte. At least 100 
leucocytes must be counted. Experience has 
shown that the largest number of cells is found 
in the edge of the smear, where the latter is 
thinnest. The number should range from two to 
eight bacteria to the cell. If more or fewer are 
present, the counting becomes more difficult in the 
one case and inaccurate in the other. The average 
number of bacteria per cell constitutes the phago- 
cytic count. The average value given by at least 
three normal individuals should be taken as the 
normal. In order to be sure that an equally thick 
emulsion of bacteria is used for each test, Bine and 
Lissner recommend that before an emulsion is 
used a count should be made with a normal ( one 's 



SERODIAGNOSTIC METHODS. 135 



own) serum. This should give always the same 
value. 

The phagocytic count of the patient's serum, 
divided by the normal count, gives the opsonic 
index of the patient. 

Slight deviations from the technique above de- 
scribed affect the result to a marked extent, and 
render the results unreliable. 

The chief use of the opsonic index in clinical 
medicine has been in the estimation of the stage 
or progress of the disease in infectious processes, 
and for the purpose of making continuous controls 
in the use of therapeutic agents, especially of 
Wright's vaccines. It may, however, serve for 
purely diagnostic purposes. Sauerbeck 61 lays 
down the following rules for use with this object 
in view : 

When the opsonic index for a certain species 
of bacteria is above normal, infection with this 
species is excluded. If the index is permanently 
lowered, the process is a local one. When it is 
permanently raised, the infection has been over- 
come, or an artificial (immunizing) infection has 
progressed to a certain point. According to 
Schottmuller and Much, 62 the opsonins exhibit a 
departure from the normal at the very beginning 
of an infection, and in infections of the lightest 

81 Neue Tatsache und Theorien der Immunitatsforschung, 
Bergman, Wiesbaden, 1907. 

62 Munch, med. Woch. (1908), No 9. 



136 SERODIAGNOSTIC METHODS. 



grade, in which agglutinin is not present in the 
serum and in which, therefore, the Widal reaction 
can be of no assistance. Since the immune opso- 
nins are more resistant to heat than the normal 
opsonins, a comparison of the index obtained be- 
fore heating with that obtained after heating for 
10 minutes at 60° may give useful results. If the 
difference is marked, it shows that the opsonins 
present are chiefly normal, and that the opsonins 
are not changed as the result of the absorption of 
bacteria. If, on the other hand, the opsonic power 
of the serum is not much reduced by heating, in- 
fection or immunization, as the case may be, may 
be inferred. Experience with this diagnostic 
method is still very limited. 

Method of Kammerer. 63 — Apparatus. — (1) 
Serum of the patient and of a healthy person, 
obtained in the usual manner, and not heated 
(not inactivated). 

(2) A bacillary emulsion in physiological salt 
solution containing 1.5 per cent, sodium citrate. 

(3) Blood of a healthy person, drawn from the 
finger-tip directly into the Wright capillary 
pipette, described above (e), p. 132. 

(4) The remaining apparatus is as described 
above under (/) to (m), p. 133. 

Technique. — Fill the capillary tube to the mark 
with blood of a normal person, then air, then blood 



63 Munch, med. Woch. (1908), No. 20. 



SERODIAGNOSTIC METHODS. 137 



again, then air, then patient's serum, then air, 
then, finally, bacillary emulsion. In a second 
capillary, blood, air, blood, air, normal serum, air, 
bacillary emulsion. 

Mixing and further handling are as described 
on p. 133 et seq. Rapid work is necessary. After 
sealing, the capillaries are placed for one-half 
hour in the incubator. Prepare the smears on 
cover-glasses and, after staining, mount in Canada 
balsam, which, on account of its greater trans- 
parency, facilitates the counting. 

The method is said to have the following 
advantages : 

(1) Simplification of the technique by combin- 
ing the separate steps of obtaining and washing 
the leucocytes. 

(2) Less injury to the leucocytes, due to wash- 
ing and centrifugating. 

(3) A relatively thick layer of leucocytes, 
which facilitates the count. 

(4) The action of the "amboceptor-like im- 
mune opsonins" is favored by the presence of 
complement, producing an increased difference 
between the normal and the patient's serum. 

The above has not been confirmed. 

Demonstration of Antistaphylolysin in Serum. 
— In patients suffering from staphylococcus 
infection. 

Principle. — One determines what amount of 



138 SERODIAGNOSTIC METHODS. 



the serum is required to protect rabbit's red blood- 
cells against solution by staphylolysin. 

Apparatus. — (a) Two or 3 c.c. defibrinated 
rabbit's blood: The blood from the severed ca- 
rotid is received in a strong, thick-walled test-tube, 
containing glass beads to about a finger's depth. 
Close the tube with a cork or rubber stopper and 
shake for several minutes. Do not fill the test-tube 
too full of blood. 

If long-eared rabbits are available, the blood 
can be obtained from the ear vein. To do this, 
rub the ear briskly with cotton wet in ether until 
the vessels become distended. Then with a sharp 
knife sever the vein transversely. As the blood 
issues from the vein it is caught in a graduated 
tube containing 2 or 3 c.c. of 1 per cent, sodium 
citrate. The blood should be well mixed with the 
citrate solution as soon as it comes in contact with 
it, to prevent coagulation. Two or 3 c.c. are thus 
collected, making the whole amount of mixture 
4 to 6 c.c. 

Eemove the citrated plasma from the cells by 
repeated washing with 10 volumes of physiological 
salt solution. After the last washing, make the 
sediment of cells up to the original volume with 
salt solution. 

(b) Staphylolysin: The most suitable medium 
for the preparation of staphylolysin is a bouillon 
containing one-third to one-half the amount of 
alkali needed to produce a distinct red reaction 



SERODIAGNOSTIC METHODS. 139 



with phenolphthalein. This amount is, of course, 
determined by titrating a small amount of the 
medium, say 10 c.c., against phenolphthalein, and 
calculating for the entire volume. One-half to one 
litre of such a bouillon is inoculated with Staphy- 
lococcus aureus, after a preliminary test has 
proved that the strain is able to produce haemol- 
ysis. This test can be made by inoculating sev- 
eral tubes containing 2 c.c. of bouillon with the 
staphylococcus strain. Beginning at the third or 
fourth day, one tube is tested each day by adding 
a drop of defibrinated rabbit's blood and placing 
for two hours in the incubator. If marked haemol- 
ysis occurs, as shown after sedimentation for 18 
hours in the ice-box, or after centrifugation, the 
strain is suitable for the purpose of the test. 

After the flask culture has grown for 13 or 14 
days, it is filtered germ-free through a Berkefeldt 
filter and 0.5 per cent, carbolic acid is added. 
Preserve in the ice-box. 

(c) The patient's serum, inactivated by heat- 
ing for one-half hour at 56°. 

(d) Physiological salt solution. 

(e) Small test-tubes, 1 cm. in diameter by 10 
cm. long. 

(/) Volumetric pipettes, 1 c.c. and 10 c.c. in 
1-10 's and 1-100 's. 

(g) Normal horse serum dried in vacuo, as 
standard serum. Dissolve fresh for each test in 
10 volumes sterile distilled water. 



140 SERODIAGNOSTIC METHODS. 



Technique. — Two separate determinations are 
involved : 

(1) The estimation of the activity of the 
staphylolysin. 

(2) The estimation of the antagonistic activity 
of the patient's serum. 

For the first, place in a series of test tubes 
the following amounts of staphylolysin : 

1.0 0.75 0.5 0.25 0.1 
and of a 10 per cent, dilution of the same, made 
with salt solution (1 c.c. lysin + 9 c.c. salt 
solution) : 

0.75 0.5 0.25 0.1 

(=0.075) (=0.05) (=0.025) (=0.01) 

and finally of a 1 per cent, dilution (1 c.c. of the 
10 per cent, dilution + 9 c.c. salt solution) : 

0.75 0.5 0.25 0.1 

( = 0.0075 ) ( = 0.005 ) ( = 0.0025 ) ( = 0.001 ) 

Make up all tubes to 2 c.c. with salt solution, add 
1 drop of rabbit 's blood, and mix. Place the tubes 
in the incubator at 37° for two hours, and, having 
noted the results, leave them over night in the ice- 
box. Eeexamine the next morning. Usually 
haemolysis is only slightly more advanced than 
after two hours in the incubator. 

The amount of staphylolysin required to effect 
complete haemolysis is made the basis of the second 
part of the test. 



SERODIAGNOSTIC METHODS. 141 



For the second portion, place the dissolving 
dose of staphylolysin as above determined in a 
series of tubes, and add the following amounts of 
the serum to be tested : 

1.0 0.75 0.5 0.25 0.1 

0.075 0.05 0.025 0.01 (added in the form of 

a 10 per cent, dilu- 
tion of the serum). 

0.0075 0.005 0.0025 0.001 (added in the form of 

a 1 per cent, dilu- 
tion of the serum). 

Bring all tubes to 2 c.c. with salt solution, add 
1 drop of rabbit 's blood, and treat as above. Note 
the volume of serum which completely neutralizes 
the hemolytic effect, as indicated by the fact that 
the supernatant fluid above the blood-cells remains 
entirely uncolored. 

In order to have a basis for comparison of the 
antihaemolytic activity of the patient's serum at 
various times, Neisser and Wechsberg recom- 
mend, in view of the possible deterioration of the 
staphylolysin, that a simultaneous determination 
of the inhibiting power of normal horse serum be 
always made, the latter to be preserved in the dry 
form, and therefore not subject to deterioration. 
The comparison of the two values (that of the 
patient's serum with that of the dry horse serum) 
gives the relative activity of the serum tested, 
as compared with the constant. This value is inde- 
pendent of the staphylolysin used. 



142 SERODIAGNOSTIC METHODS. 



According to Arndt 64 and others, a high anti- 
staphylolysin content of the serum is always a 
sign of staphylococcns infection, even if it is im- 
possible to establish fixed limits for the values. 

Determination of Diphtheria Toxin in the Blood 
Serum (Uffenheimer's Method). 65 — Principle. — 
Small amounts of diphtheria toxin, too slight to 
cause death of a laboratory animal (guinea-pig), 
may, nevertheless, cause, after 24 hours, distinct 
cedema or infiltration at the seat of injection into 
the same animal. 

Apparatus. — (a) 0.1 to 0.3 c.c. serum from the 
patient. 

(b) A guinea-pig weighing about 250 grammes. 

(c) Physiological salt solution, to dilute the 
serum. 

(d) A sterilizable syringe of 2 to 4 c.c. 
capacity. 

(e) A sterile sedimentation glass, in which to 
mix the serum and salt solution. 

(/) Operating table and instruments. 

Technique. — The serum to be injected is made 
up to 2 to 4 c.c. with salt solution, and injected into 
the guinea-pig intraperitoneally {not intramuscu- 
larly!). After 17 to 20 hours one can feel a very 
distinct doughy cedema of the abdominal cavity. 
At the end of 48 hours kill the animal by a blow on 



^Deutsch. med. Woch., 1907. 

"'Munch, med. Woch. 3 (1906), and (1907), No. 3. 



SERODIAGNOSTIC METHODS. 143 



the back of the neck, place it on the operating 
table, and open the thorax and abdomen, The 
oedema can then be seen readily. 

The diagnostic value of this method has not yet 
been adequately established. On the one hand, 
Uffenheimer maintains that the diagnosis of true 
diphtheria can often be made more quickly by this 
method than by bacteriological examination; on 
the other hand, it is true that the reaction is not 
always present in known diphtheria. 



INDEX 



Agglutination reaction, 41 
macroscopic method, 42 
microscopic method, 45 
Alexin, see Complement 
Ankylostomiasis, meiostagmin 

reaction in, 120 
Antihaemolytic reactions, 75 
Antistaphylolysin, estimation of, 
137 

Bacteria, agglutination for recog- 
nition of, 41 
Pfeiffer phenomenon for rec- 
ognition of, 55 
Blood-cells, washing of, 12, 77 
Blood, animal, obtaining large 
amounts, 4, 5, 6 
obtaining small amounts, 
2 

defibrinating, 11 
forensic determination of, 
complement fixation 
method, 75 
human, obtaining large 
amount, 10, 11 
obtaining small amount, 
7,9 

precipitin method, 22 
Blood-serum, diphtheria toxin 
in, 142 

preservation of, with anti- 
septics, 13, 15 
by drying, 19 



Blood-serum, preservation of, 
by freezing, 18 
by germ free filtration, 15 
in living animal, 21 
with sodium sulphate, 21 

Cancer, diagnosis of, antitrypsin 
reaction, Bergmann and 
Meyer, 117 

antitrypsin reaction, Mar- 
cus, 115 

cobra venom, reaction of, 
113 

complement fixation in, 
101 

cytolytic reaction, 64 
meiostagmin reaction, 120 
Cholera, agglutination reaction 
in, 46, 52 
Pfeiffer phenomenon in, 59, 
60 

Cobra venom reactions, 109 
Complement, obtaining, 76 

estimation of, in patient's 
serum, 72 

Diphtheria, toxin in serum, 142 

Ecchinococcus, meiostagmin re- 
action in, 120 

Gonorrhoea, diagnosis of, by 
complement fixation, 104 
145 



146 



INDEX 



Infection, mixed, diagnosis of, 
method of Castellani, 53 

Lues, see Syphilis 

Meat adulteration, complement 
fixation method, 75 
precipitin method, 28 
Meiostagmin reaction, 119 

Opsonins, estimation of, Bine 
and Lissner, 128 
Kammerer, 136 

Paratyphoid, agglutination re- 
action in, 46, 52 
Pfeiffer phenomenon in, 59, 
60 

Pfeiffer phenomenon, 55 
Pregnancy, diagnosis of, 68 
Psychoreaction of Much and 
Holzmann, 109 

Rabbits, injection of, intraperi- 
toneally, 1 
intravenously, 2 

Sarcoma, see Cancer 
Serum, see Blood-serum 
Staphylococcus infection, anti- 

staphylolysin in, 137 
Syphilis, cobra venom reaction 
of, 111 

complement fixation, Bauer, 
96 

Hoehne, 93 



Syphilis, complement fixation, 
Landsteiner, Muller, 
and Potzl, 100 

Stern, 94 

Taege, 89 

Tschernoguboff, 98 

Wassermann and Me- 
ier, 85 

floccule forming reactions, 
Elias, Neubauer, 
Porges, Salomon, 
38 

Herman and Perutz, 
40 

Klausner, 39 

Porges and Meier, 37 
precipitin reaction, Fornet 
and Schereschewsky, 35 
meiostagmin reaction, 120 

Tuberculosis, complement fixa- 
tion, 107 
meiostagmin reaction, 120 

Tumor, see Cancer 

Typhoid, agglutination reaction, 
46, 52 

complement fixation, 75 
meiostagmin reaction, 120 
Pfeiffer phenomenon, 59, 60 

Wassermann reaction, methods 
for, 85 

Widal reaction, Ficker method, 
46 

macroscopic method, 46 
microscopic method, 52 



MAY I i9is 



